Charge-reversed n-terminal spider silk protein domain and uses thereof

ABSTRACT

A protein comprising a moiety of 100-160 amino acid residues having at least 70% identity with the N-terminal (NT) fragment of a spider silk protein, wherein the amino acid residue corresponding to position 40 in NT is selected from the group consisting of Lys, Arg and His; and wherein the amino acid residue corresponding to position 65 in NT is selected from the group consisting of Asp and Glu, is useful as a moiety in a fusion protein for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 15/544,945, filed on Jul. 20, 2017, which is the National Phase under 35 U.S.C. § 371 of International Application No. PCT/EP2016/077415, filed on Nov. 11, 2016, which claims the benefit under 35 U.S.C. § 119(a) to patent application Ser. No. 15194623.3, filed in European Patent Office on Nov. 13, 2015, Patent application Ser. No. 16186679.3, filed in European Patent Office on Aug. 31, 2016, and patent application Ser. No. 16193082.1, filed in European Patent Office on Oct. 10, 2016, all of which are hereby expressly incorporated by reference into the present application.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of proteins and polypeptides, and more specifically to expression and production of spider silk proteins (spidroins) and other, non-spidroin proteins and polypeptides. The present invention provides novel proteins which are useful in themselves and as a moiety in novel fusion proteins for expression and production of the desired proteins and polypeptides, as well as nucleic acid molecules encoding these novel proteins and fusion proteins. The present invention also provides a method of expressing and producing a desired protein or polypeptide.

BACKGROUND TO THE INVENTION

Production of proteins and polypeptides from DNA can be achieved in various hosts, but a common problem is the formation of insoluble protein/polypeptide aggregates. This may severely impede or even prevent production of a functional protein/polypeptide. The problem is typically aggravated with low-solubility proteins and polypeptides, e.g. membrane-associated proteins and polypeptides.

Membrane-associated proteins account for 20-30% of the proteome of the cell and are the targets of many currently available pharmaceutical drugs. In order to get inserted into the membrane, the protein needs at least one stretch of 15-20 amino acid residues that, according to the biological hydrophobicity scale, promotes membrane insertion. At the same time, hydrophobicity of the amino acid side chains is an important determinant of aggregation potential, and hydrophobic amino acid residues (Val, Ile, Phe and Cys) promote β-sheet formation and are overrepresented in amyloid forming core regions of many disease associated proteins. Accordingly, membrane associated proteins are prone to aggregate, which may severely impede or even prevent the production of a functional recombinant protein.

For instance, lung surfactant protein C (SP-C) is a transmembrane (TM) protein that is difficult to produce recombinantly because of its extremely hydrophobic nature. SP-C is produced by alveolar type II cells and is a constituent of surfactant, that is necessary to prevent alveolar collapse at end expiration. Neonatals often suffer from respiratory distress due to insufficient amounts of surfactant. Today, this condition is treated with surfactant preparations extracted from animal lungs, e.g. Curosurf®, Infasurf®, Alveofact® and Survanta®. Treatment with exogenous surfactant is also potentially beneficial for adult patients with respiratory distress, but the supply of surfactant is too limited and the price very high. Surfactant preparations based on peptides produced in a heterologous system would be superior to the natural extracts used today (and formulations containing chemically synthesized peptides) due to lower production cost and higher production volume. It would also be favourable from a regulatory point of view.

SP-C33Leu is a variant of SP-C, where the N-terminal part is truncated with two residues, two Cys residues are replaced with Ser, one Leu residue is replaced with Lys, and one Met residue is replaced with Leu, and the residues spanning the membrane (normally mainly Val) are exchanged for Leu in order to enhance the stability of the transmembrane helix. KL4 is another surfactant analogue designed to imitate the properties of the lung surfactant protein B (SP-B) and consists of iterated repeats of Lys-Leu-Leu-Leu-Leu. SP-C33Leu and KL4 recapitulate the function of native surfactant peptides, including transmembraneous insertion, but are less prone to aggregate and may therefore be feasible to produce in large quantities for development of a synthetic surfactant preparation. Both peptides can be produced by chemical synthesis but the cost is considerable and the process renders bi-products that may be difficult to remove and to characterize.

The pulmonary surfactant proteins A (SP-A) and D (SP-D) do not insert into membranes but rather play a role in the pulmonary immune response through their carbohydrate-binding domains. They are large water-soluble protein complexes involved in the first line defence of the lungs and regulate the functions of the innate immune cells (e.g. macrophages) as well as the adaptive immune cells. The proteins belong to the collectin family of C-type lectins composed of an N-terminal collagen-like region and a C-terminal calcium-dependent carbohydrate recognition domain. In their functional form, the proteins are arranged as trimeric polypeptide chains via their N-terminal regions and further assemble into larger oligomers of different shapes. SP-A consists of six trimeric subunits arranged as a “bouquet”, while SP-D arrange as a cruciform of four trimeric subunits. Although the proteins are hydrophilic, they are reluctant to recombinant production and have so far been expressed as insoluble inclusion bodies and purified by denaturation and refolding. Currently, surfactant preparations in clinical use do not contain SP-A or SP-D and there is an interest to investigate if current surfactant therapies could be improved by adding these components that are a natural part of surfactants. Human SP-A and SP-D can be isolated from patients with alveolar proteinosis or from amniotic fluid but the yields are low and the oligomeric state is non-uniform. Recombinant production of the proteins would allow for scaled-up and reproducible manufacturing for therapeutic use but so far the attempts have been unconvincing.

Other examples of proteins and polypeptides that pose difficulties when expressed from recombinant DNA are Aβ-peptide, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF and actin; SP-B, α-defensins and β-defensins; class A-H apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; avidin and streptavidin; protease 3C; and immunoglobulins and fragments thereof.

One solution to this problem is to express the desired protein or polypeptide as a fusion protein with a solubility enhancing peptide/domain, i.e. a protein or polypeptide that provides the required solubility. The fusion protein may be cleaved, and the desired protein isolated. Alternatively, the desired protein/polypeptide may be maintained integrated in the soluble fusion protein, where it remains functional and can be subjected to further characterization, e.g. activity and interaction studies, structure determination and crystallization. Thioredoxin (Trx) is among the most widely used solubility enhancing fusion partners that accumulate to high levels in the E. coli cytoplasm and has proven to dramatically increase the solubility of many heterologous proteins and small peptides. Another successful fusion partner is the immunoglobulin binding domain B1 from Streptococcal protein G (PGB1). The high stability and small size (56 residues) of this domain gives it exceptional qualities for expression of small domains and peptides and for downstream structural characterization.

WO 2011/115538 discloses a fusion protein comprising a solubility-enhancing moiety which is derived from the N-terminal (NT) fragment of a spider silk protein and a moiety which is a desired protein or polypeptide. A pH above 6.4 is preferred to prevent assembly of the solubility-enhancing moiety.

EP 2 644 619 A1 also discloses a fusion protein comprising a solubility-enhancing moiety which is derived from the N-terminal (NT) fragment of a spider silk protein and a moiety which is a desired protein or polypeptide. The solubility-enhancing moiety is a constitutive monomer also below a pH of 6.4, but does not increase expression levels of the resulting fusion proteins compared to the wildtype NT fragment.

Despite these progresses in the field, the fusion protein approach has limitations in terms of expression, stability and solubility of the product. The use of fusion partners in large-scale heterologous protein production is uncommon, mainly due to the need of additional expensive chromatographic steps and/or difficulties in removing the fusion partner.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a fusion partner, i.e. a moiety in a fusion protein, which is useful for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide.

It is also an object to provide novel proteins and a method for improved recombinant production method for hydrophobic proteins and polypeptides and proteins and polypeptides with hydrophobic regions.

It is a further object of the present invention to provide a fusion partner which is a stable monomer over a broad pH range.

It is another object of the present invention to provide a simplified method of producing and isolating a desired protein or polypeptide involving expression in a fusion protein.

For these and other objects that will be evident from the following specification, the present invention provides according to a first aspect a protein according to the appended claims. The present invention is generally based on the discovery that this charge-reversed NT mutant is unable to dimerize, stabilized, hypersoluble and enables efficient production of transmembrane and aggregation-prone proteins. Fusion proteins according to the invention can advantageously form micelles. The present invention provides according to a second aspect use of this protein as a moiety in a fusion protein for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide.

The present invention provides according to a third aspect a method of producing a desired protein or polypeptide according to the appended claims.

List of appended sequences SEQ ID NO 1 NT_(D40K/R/H, K65D/E) 2 NT_(D40K, K65D) 3 NT_(D40R, K65D) 4 NT_(D40H, K65D) 5 NT_(D40K, K65E) 6 NT_(D40R, K65E) 7 NT_(D40H, K65E) 8 NT_(D40K, K65D) (DNA) 9 NT full-length 10 consensus NT sequence 11 NT_(wt) 12 NT_(wt) (DNA) 13 NT Euprosthenops australis MaSp1 14 NT Latrodectus geometricus MaSp1 15 NT Latrodectus hesperus MaSp1 16 NT Nephila clavipes MaSp1 17 NT Argiope trifasciata MaSp2 18 NT Latrodectus geometricus MaSp2 19 NT Latrodectus hesperus MaSp2 20 NT Nephila inaurata madagascariensis MaSp2 21 NT Nephila clavipes MaSp2 22 NT Argiope bruennichi cylindriform spidroin 1 23 NT Nephila clavata cylindriform spidroin 1 24 NT Latrodectus hesperus tubuliform spidroin 25 NT Nephila clavipes flagelliform silk protein 26 NT Nephila inaurata madagascariensis flagelliform silk protein 27 β17 28 Human SP-B 29 Mouse SP-B 30 Pig SP-B 31 Rabbit SP-B 32 Rat SP-B 33 Mini-B 34 Mini-BLeu 35 Mini-B27 36 1a AA 37 1b AA 38 1a LL 39 1b LL 40 SP-C 41 SP-C(Leu) 42 SP-C33 43 SP-C30 44 SP-C33(Leu) 45 LL-37 46 KL4 47 hSP-A1₈₁₋₂₂₈ 48 hSP-A2₈₁₋₂₂₈ 49 hSP-D₂₀₄₋₃₅₅ 50 Bri2₁₁₃₋₂₃₁ (Bri2-Brichos) 51 Neuroserpin 52 0 AAAA 53 0 LLLL 54 Green Fluorescent Protein (GFP) 55 eGFP 56 NT_(D40K, K65D)-SP-C33Leu 57 NT_(wt)-SP-C33Leu 58 NT_(D40K, K65D)-SP-C33Leu (DNA) 59 NT_(wt)-SP-C33Leu (DNA) 60 NT_(D40K, K65D)-KL4 61 NT_(wt)-KL4 62 NT_(D40K, K65D)-KL4 (DNA) 63 NT_(wt)-KL4 (DNA) 64 NT_(D40K, K65D)-β17 65 NT_(D40K, K65D)-β17 (DNA) 66 NT_(D40K, K65D)-Bri2₁₁₃₋₂₃₁ 67 NT_(wt)-Bri2₁₁₃₋₂₃₁ 68 NT_(D40K, K65D)-Bri2₁₁₃₋₂₃₁ (DNA) 69 NT_(wt)-Bri2₁₁₃₋₂₃₁ (DNA) 70 NT_(D40K, K65D)-hSP-A1 71 NT_(D40K, K65D)-hSP-A2 72 NT_(D40K, K65D)-hSP-D 73 NT_(D40K, K65D)-hSP-A1₈₁₋₂₂₈ 74 NT_(D40K, K65D)-hSP-A2₈₁₋₂₂₈ 75 NT_(D40K, K65D)-hSP-D₂₀₄₋₃₅₅ 76 NT_(D40K, K65D)-hSP-A1 (DNA) 77 NT_(D40K, K65D)-hSP-A2 (DNA) 78 NT_(D40K, K65D)-hSP-D (DNA) 79 NT_(D40K, K65D)-hSP-A1₈₁₋₂₂₈ (DNA) 80 NT_(D40K, K65D)-hSP-A2₈₁₋₂₂₈ (DNA) 81 NT_(D40K, K65D)-hSP-D₂₀₄₋₃₅₅ (DNA) 82 NT_(D40K, K65D)-Aβ₁₋₄₂ 83 NT_(D40K, K65D)-Aβ₁₋₄₂ (DNA) 84 NT_(D40K, K65D)-hIAPP 85 NT_(D40K, K65D)-hIAPP (DNA) 86 NT_(wt)-hCAP18 87 NT_(D40K, K65D)-hCAP18 88 NT_(wt)-hCAP18 (DNA) 89 NT_(D40K, K65D)-hCAP18 (DNA) 90 NT_(D40K, K65D)-nicastrin 91 NT_(D40K, K65D)-nicastrin (DNA) 92 NT_(wt)-eGFP 93 NT_(D40K, K65D)-eGFP 94 NT_(wt)-eGFP (DNA) 95 NT_(D40K, K65D)-eGFP (DNA) 96 NT Araneus ventricosus MiSp 97 NT Latrodectus hesperus MiSp 98 NT Uloborus diversus MiSp 99 NT Metepeira grandiosa MiSp 100 NT_(A72R)-SP-C33Leu 101 NT_(D40K, K65D)-FN_(cc)-4Rep-CT_(MiSp) 102 Z-FN_(cc)-4Rep-CT_(MiSp) 103 NT_(D40K, K65D)-FN_(cc)-4Rep-CT_(MaSp) 104 Z-FN_(cc)-4Rep-CT_(MaSp) 105 NT_(D40K, K65D)-sCD40-4Rep-CT_(MaSp) 106 Z-sCD40-4Rep-CT_(MaSp) 107 NT_(D40K, K65D)-sCD40-sortase 108 Z-sCD40-sortase

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment of spidroin N-terminal domains.

FIG. 2 shows the ratio of fluorescence emission at 339 nm and 351 nm vs pH for NT_(D40K/K65D) and wild type NT.

FIG. 3 is a comparison of NT_(wt) and NT_(D40K/K65D) using 2D HSQC NMR.

FIG. 4 shows the urea-induced denaturation of NT_(wt) and NT_(D40K/K65D) as a measure of stability.

FIG. 5 shows thermal stability of NT_(wt) and NT_(D40K/K65D) measured with CD spectroscopy.

FIG. 6 shows refolding capacity of NT_(wt) and NT_(D40K/K65D) measured with CD spectroscopy.

FIG. 7-8 are SDS-PAGE evaluations of fusion proteins and resulting purified proteins and peptides.

FIG. 9 shows ESI-MS spectra of SP-C33Leu produced by the inventive method.

FIG. 10 illustrates the effects of rSP-C33Leu on tidal volumes.

FIG. 11 illustrates the effects of rSP-C33Leu on lung gas volumes.

FIG. 12 is an SDS-PAGE evaluation of β17 fusion proteins and resulting purified polypeptide.

FIG. 13 shows gel filtration of soluble NT_(D40K/K65D)-β17 fusion protein.

FIG. 14 is a graph of ThT fluorescence illustrating fibrillation of β17 after proteolysis of the fusion protein with thrombin.

FIG. 15 is an SDS-PAGE evaluation of expression and solubility of Bri2 BRICHOS fusion proteins.

FIG. 16 is an SDS-PAGE analysis of Bri2 BRICHOS purification.

FIG. 17 shows gel filtration to determine the oligomeric state of Bri2₁₁₃₋₂₃₁ fusion proteins.

FIG. 18 shows a SDS-PAGE analysis of expressed hCAP18 in fusion with NT_(wt) or NT_(D40K/K65D).

FIG. 19 shows SEC chromatograms of NT_(D40K/K65D)-SP-C33Leu fusion proteins.

FIG. 20 shows TEM micrographs of negatively stained proteins containing NT_(D40K/K65D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with production and expression of proteins and polypeptides. Depending on the purpose with this production, the end product may vary. It may for instance be desirable to obtain the desired protein or polypeptide inserted in a lipid membrane, in solution or associated with other biomolecules. It shall also be realized that it may also be highly desirable to obtain the desired protein or polypeptide as part of a fusion protein, which may provide a suitable handle for purification and detection and/or provide desirable properties, e.g. stability and solubility. Maintaining the desired protein or polypeptide functionally integrated in a soluble fusion protein is useful to characterize and study the desired protein or polypeptide.

The present invention is generally based on the insight of the usefulness of a specific variant of the N-terminal (NT) fragment of a spider silk protein due to its surprising capacity to be present as a soluble monomer regardless of the pH of the surrounding aqueous medium, and its excellent properties as a fusion partner due to its extraordinary high solubility.

Spider silk consists mainly of large and aggregation-prone proteins (spidroins) that are produced in abdominal glands of spiders. They are built up from extensive and mainly hydrophobic stretches of repeated alanine- and glycine-rich segments flanked by globular and hydrophilic domains at the N-terminal (NT) and C-terminal (CT) end. During spinning, spidroins are passaged through a narrowing duct and convert from soluble protein into solid fibers in a process that involve precise control of the environmental conditions including level of hydration, shear forces, ion composition, pH and carbon dioxide pressure. Despite their aggregation-prone nature, spidroins are stored at a remarkably high concentration (30-50% w/w) in the spider silk gland.

The NT domain is the most conserved part of spidroins and folds into a soluble ˜130 residue 5-helix bundle with a dipolar charge distribution. Furthermore, NT has an important role in fiber formation as it forms anti-parallel dimers at pH below 6.5, which is considered to be a critical step for interconnecting spidroin micelles in the spinning duct.

In known spider MaSp1 and MaSp2 silk protein species (see e.g. FIG. 1), D40 and K65 of the NT moiety (SEQ ID NO: 9) are conserved and mediate intersubunit electrostatic interactions by salt bridges between the side chains of the negatively charged D40 and the positively charged K65, which stabilizes the dimer configuration. Similarly, there are corresponding oppositely charged amino acid residues in known minor ampullate spider silk (MiSp) species. It has now been realized and demonstrated in the Examples that charge-reversing mutations of these residues to provide a positively charged (basic) amino acid residue in position 40 and a negatively charged (acidic) amino acid residue in position 65 surprisingly abolish the intersubunit salt bridges between these positions, which in turn has a critical impact on the pH-dependent dimerization capacity of the NT fragment. As a result of these charge-reversing mutations, the NT mutant surprisingly becomes a stable monomer over a broad pH interval and attains excellent properties as a fusion partner, including high expression, high stability and pH insensitivity of the resulting fusion protein together with the desired protein or polypeptide. The NT mutant is highly soluble in water. The high expression levels of the resulting fusion proteins using the charge-reversed NT mutant according to the present invention are particularly surprising considering that another known NT variant, NT_(A72R), achieves lower expression levels than NT_(wt) of the corresponding fusion proteins. Without desiring to be bound to any particular scientific theory, it is suggested that the improved stability and refolding capacity of the charge-reversed mutant NT according to the present invention can be explained by the less dipolar charge distribution that reduces destabilizing charge clusters.

The yields of soluble fusion proteins after expression in E. coli are typically at least 2 times higher compared to conventional tags like thioredoxin and PGB1, and the fusion proteins can be purified to homogeneity, e.g. by salt precipitation and/or precipitation in an organic solvent. The cleaved off desired proteins or peptides are active, e.g. in an animal model of disease. The charge reversed NT mutant is unable to dimerize, stabilized, hypersoluble, yields higher amounts of fusion proteins, and enables production of aggregation-prone proteins that have previously been refractory to recombinant production.

Without desiring to be limited to any specific theory, the experimental results disclosed herein support that recombinant production of desired peptides or proteins can be enhanced using the charge-reversed NT mutant according to the invention as a fusion tag that mediates solubility and shields hydrophobic regions of the desired protein from the aqueous surrounding within micelle-like particles. The mutant NT_(D40K/K65D) is unable to dimerize at low pH due to a reduced dipolar charge distribution and is therefore able to mediate solubility in a wider pH range. Interestingly, size exclusion chromatography of NT_(D40K/K65D)-SP-C33Leu (FIG. 19) shows that that the purified amphipathic fusion protein arrange into 510 kDa assemblies and micelle-like particles around 10-15 nm in size are observed by TEM (FIG. 20).

It is therefore considered that the formation of micelles, or micelle-like particles, comprising or consisting of fusion proteins comprising the charge-reversed NT mutant according to the invention are a useful intermediate structure in the recombinant production of desirable proteins and polypeptides, in particular of hydrophobic proteins and polypeptides and proteins and polypeptides with hydrophobic regions, e.g. membrane proteins and polypeptides and membrane-associated proteins and polypeptides. The micelles typically have a size in the range of 5-100 nm, such as 5-30 nm, and preferably 5-20 or 10-15 nm.

Membrane-associated proteins and numerous other commercially relevant proteins are difficult to produce and purify in their native form due to their hydrophobic and aggregation-prone nature. The lack of sustainable and generic production regimes makes these proteins difficult to manufacture and evaluate, e.g. as pharmaceuticals. The charge-reversed NT mutant according to the invention without the ability to dimerize demonstrated an improved stability and refolding capacity and was used as a highly soluble fusion partner for production of surfactant peptide analogues SP-C33Leu, KL4, surfactant proteins SP-A and SP-D and truncated variants thereof, the amyloid-forming polypeptides β17, Aβ and IAPP, hCAP18, nicastrin, eGFP and the Bri2-BRICHOS protein domain. The obtained amounts of fusion proteins were up to 8-fold higher compared to PGB1 fusion proteins, and all peptides/proteins could be produced as soluble and functional protein after removal of the NT domain.

The use of fusion partners in large-scale heterologous protein production is uncommon, mainly due to the need of additional expensive chromatographic steps and/or difficulties in removing the fusion partner. However, NT and charge-reversed NT allow for efficient purification of hydrophobic target peptides using simple NaCl precipitation and an ethanol extraction step that circumvents the need for chromatography. The procedure described herein represents a cheap, efficient and, from a regulatory point of view, beneficial way of producing proteins, e.g. non-animal derived SP-C33Leu for clinical use in synthetic lung surfactants. It is shown herein that recombinant SP-C33Leu is identical to the synthetic peptide and a mixture of recombinant SP-C33Leu and synthetic phospholipids is similar to the porcine derived surfactant Curosurf® in terms of function in an animal model of respiratory distress.

The surfactant proteins SP-A and SP-D do not insert into membranes but rather play a role in the pulmonary immune response through their carbohydrate-binding domains. Although the proteins are hydrophilic, they are reluctant to recombinant production. Using charge-reversed NT as a fusion partner enables high expression and subsequent purification of soluble derivatives of SP-A and SP-D. This shows that charge-reversed NT works as a general solubility-enhancing fusion partner for biotechnical applications, enabling heterologous production of TM peptides as well as proteins that today are difficult to produce due to their hydrophobicity or aggregation propensity.

Thus, the present invention provides according to a first aspect a protein comprising a moiety of 100-160 amino acid residues having at least 70% identity with SEQ ID NO: 1 or SEQ ID NO: 96, wherein the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is a basic amino residue, selected from the group consisting of Lys, Arg and His; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is an acidic amino acid residue, selected from the group consisting of Asp and Glu.

Wildtype NT is highly water-soluble and useful e.g. as a solubility-increasing moiety in a fusion protein for the expression of a desired protein or polypeptide, but it also form dimers at a pH interval of 5.5-7.2 which increases the risk of undesirable aggregation of the fusion proteins. This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptides. It is also a useful pH interval for certain purification protocols, e.g. when using ion exchange, such as cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) as a purification principle. It is also a useful pH interval for certain expression hosts, e.g. yeasts. It has now been realized that the charge-reversed double mutant NT according to the invention decreases the capacity of the protein to form dimers, without adversely affecting its solubility or its capacity to increase the solubility of a desired protein or polypeptide when they are linked in a fusion protein. The mutant NT protein according to the invention is therefore useful in itself to study the physiologically relevant NT monomer as such. The mutant NT protein according to the invention is also useful as a solubility-increasing moiety in a fusion protein, since it decreases the risk of undesirable aggregation of the fusion proteins, and thereby opens up a new pH window (5.5-7.2) in which charge-reversed double mutant NT from spider silk protein can be used in biochemical applications when solubility of protein/polypeptide monomers in aqueous solutions is desirable, e.g. in production or characterization of desirable proteins or polypeptides. It has surprisingly been determined from experimental data that although these changes in positions 40 and 65 do not alter the net charge of the resulting protein, its capacity to provide stability and solubility to any desirable protein/polypeptide moiety to which it is fused is improved.

In a preferred embodiment, the fusion protein according to the invention is present as micelles or micelle-like particles. The micelles typically have a size in the range of 5-100 nm, such as 5-30 nm, 5-20 nm, or 10-15 nm. This intermediate micellar structure is believed to support the high stability and solubility, and ultimately the high yield, of the fusion proteins, and in particular of fusion proteins comprising hydrophobic proteins and polypeptides as well as proteins and polypeptides with hydrophobic regions, thus protecting the water-insoluble peptide during expression and purification in aqueous solvents. The micelles typically have a size in the range of 5-100 nm, such as 5-30 nm, and preferably 5-20 nm or 10-15 nm.

In a preferred embodiment, the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is Lys or Arg, and preferably Lys. In one preferred embodiment, the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is Asp. The six possible and preferred variants regarding these two positions are presented as SEQ ID NO: 2-7. A particularly preferred variant is SEQ ID NO: 2, wherein the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is Lys; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is Asp. For avoidance of doubt, the corresponding mutated positions in SEQ NO: 2-7 are positions 36 and 61 due to a shorter N-terminal end.

In preferred embodiments, the amino acid residue corresponding to position 72 in SEQ ID NO: 1 is not Arg. It is preferably a non-charged residue, i.e. not Lys, Arg, His, Glu or Asp. In certain preferred embodiments, the amino acid residue corresponding to position 72 in SEQ ID NO: 1 is selected from the group consisting of Ala, Val, Phe, Pro, Leu, Ile, Trp, Met, Cys and Gly; and preferably Ala, Val, Leu, Ile and Gly. In a preferred embodiment, the amino acid residue corresponding to position 72 in SEQ ID NO: 1 is Ala or Gly, preferably Ala.

As set out above, the inventive moiety is derived from the NT fragment of a spider silk protein, or spidroin. Although the examples by necessity relate to specific NT fragments, in this case proteins derived from major spidroin 1 (MaSp1) from Euprosthenops australis, it is considered that the method disclosed herein is applicable to any similar protein moiety. The terms “spidroins” and “spider silk proteins” are used interchangeably throughout the description and encompass all known spider silk proteins, including major ampullate spider silk proteins which typically are abbreviated “MaSp”, or “ADF” in the case of Araneus diadematus, and minor ampullate spider silk proteins, typically abbreviated “MiSp”. The major ampullate spider silk proteins are generally of two types, 1 and 2. These terms furthermore include the new NT protein fragments according to the invention, as defined in the appended claims and itemized embodiments, and other non-natural proteins with a high degree of identity and/or similarity to the known spider silk NT protein fragments.

The inventive moiety has a high degree of similarity to the N-terminal (NT) amino acid sequence of spider silk proteins. As shown in FIG. 1, these amino acid sequences are well conserved among various species and spider silk proteins, including MaSp1, MaSp2 and MiSp. The skilled person is therefore well aware how, and to what extent, the amino acid sequence may be varied without departing from the properties and functionality of the N-terminal spider silk protein fragment. Table 1 lists the spidroin NT fragments which are aligned in FIG. 1 and other inventive NT moieties, denoted with GenBank accession entries.

TABLE 1 Spidroin NT fragments GenBank SEQ Code Species and spidroin protein acc. no. ID NO Ea MaSp1 Euprosthenops australis MaSp 1 AM259067 13 Lg MaSp1 Latrodectus geometricus MaSp 1 ABY67420 14 Lh MaSp1 Latrodectus hesperus MaSp 1 ABY67414 15 Nc MaSp1 Nephila clavipes MaSp 1 ACF19411 16 At MaSp2 Argiope trifasciata MaSp 2 AAZ15371 17 Lg MaSp2 Latrodectus geometricus MaSp 2 ABY67417 18 Lh MaSp2 Latrodectus hesperus MaSp 2 ABR68855 19 Nim MaSp2 Nephila inaurata madagascariensis AAZ15322 20 MaSp 2 Nc MaSp2 Nephila clavipes MaSp 2 ACF19413 21 Ab CySp1 Argiope bruennichi cylindriform BAE86855 22 spidroin 1 Ncl CySp1 Nephila clavata cylindriform BAE54451 23 spidroin 1 Lh TuSp1 Latrodectus hesperus tubuliform ABD24296 24 spidroin Nc Flag Nephila clavipes flagelliform silk AF027972 25 protein Nim Flag Nephila inaurata madagascariensis AF218623 26 flagelliform silk protein (translated) Av MiSp Araneus ventricosus MiSp AFV31615 96 Lh MiSp Latrodectus hesperus MiSp ADM14321 97 Ud MiSp Uloborus diversus MiSp ADM14326 98 Mg MiSp Metepeira grandiosa MiSp ADM14328 99

Only the part corresponding to the N-terminal domain is shown for each sequence, omitting the signal peptide. Nc flag and Nlm flag are translated and edited according to Rising A. et al. Biomacromolecules 7, 3120-3124 (2006)).

It is not critical which specific NT moiety is present in the proteins according to the invention, as long as the NT moiety is not entirely missing. Thus, the NT moiety according to the invention can be selected from any of the MaSp1 or MaSp2 amino acid sequences shown in FIG. 1 or sequences with a high degree of similarity or MiSp amino acid sequences, such as SEQ ID NO: 96-99. A wide variety of sequences can be used in the fusion protein according to the invention. Based on the homologous sequences of FIG. 1, the following sequence constitutes a consensus MaSp NT amino acid sequence:

(SEQ ID NO: 10) QANTPWSSPNLADAFINSF(M/L)SA(A/I)SSSGAFSADQLDDMSTIG (D/N/Q)TLMSAMD(N/S/K)MGRSG(K/R)STKSKLQALNMAFASSMA EIAAAESGG(G/Q)SVGVKTNAISDALSSAFYQTTGSVNPQFV(N/S)E IRSLI(G/N)M(F/L)(A/S)QASANEV.

The sequence of the inventive moiety according to the invention has preferably at least 50% identity, preferably at least 60% identity, to the consensus amino acid sequence SEQ ID NO: 10, which is based on the wildtype NT amino acid sequences of FIG. 1. In a preferred embodiment, the sequence of the inventive moiety according to the invention has at least 65% identity, preferably at least 70% identity, to the consensus amino acid sequence SEQ ID NO: 10. In preferred embodiments, the solubility-enhancing moiety according to the invention has furthermore 70%, preferably 80%, similarity to the consensus amino acid sequence SEQ ID NO: 10.

A representative inventive moiety according to the invention is SEQ ID NO: 2 (encoded by SEQ ID NO: 8), which is derived from the Euprosthenops australis NT moiety (SEQ ID NO: 9) with replacement of aspartic acid in position 40 with lysine or any other basic residue, and with replacement of lysine in position 65 with aspartic acid or any other acidic residue, as set out hereinabove and in SEQ ID NO: 1. According to a preferred embodiment of the invention, the inventive moiety furthermore has at least 70% identity, such as at least 75% identity, preferably at least 80% identity to SEQ ID NO: 1 or any of SEQ ID NO: 96-99 or any individual amino acid sequence in FIG. 1. In preferred embodiments of the invention, the inventive moiety has at least 85%, such as at least 90% or even 95% identity, to SEQ ID NO: 1: or any of SEQ ID NO: 96-99 or any individual amino acid sequence in FIG. 1. In preferred embodiments of the invention, the solubility-enhancing moiety is identical to SEQ ID NO: 9 or any of SEQ ID NO: 96-99 or any individual amino acid sequence in FIG. 1, with the proviso that acidic residue in position 40 and the basic residue in position 65 (or the corresponding positions as it may be) are replaced as set out hereinabove.

The term “% identity”, as used throughout the specification and the appended claims, is calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson, J. D., Higgins, D. G. and Gibson, T. J., Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.

The term “% similarity”, as used throughout the specification and the appended claims, is calculated as described for “% identity”, with the exception that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys are similar; the basic residues Lys, Arg and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gln, Asn, Ser, Thr and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

Throughout this description, alternative embodiments according to the invention fulfill, instead of the specified percentage of identity, the corresponding percentage of similarity. Other alternative embodiments fulfill the specified percentage of identity as well as another, higher percentage of similarity, selected from the group of preferred percentages of identity for each sequence. For example, a sequence may be 70% similar to another sequence; or it may be 70% identical to another sequence; or it may be 70% identical and 90% similar to another sequence.

The inventive moiety contains from 100 to 160 amino acid residues. It is preferred that the inventive moiety contains at least 100, or more than 110, preferably more than 120, amino acid residues. It is also preferred that the inventive moiety contains at most 160, or less than 140 amino acid residues. A typical inventive moiety contains approximately 130-140 amino acid residues.

As set out in detail in WO 2011/115538 which is incorporated in its entirety herein, the N-terminal (NT) fragment of a spider silk protein is particularly useful as a solubility-enhancing moiety in a fusion protein that is produced from recombinant DNA. According to a further aspect, the present invention is further based on the insight of the usefulness of the charge-reversed double mutant NT according to the invention in such a fusion protein due to its capacity to be present as a soluble monomer regardless of the pH of the surrounding aqueous medium. The resulting fusion protein is surprisingly stable and can be produced in high yield.

According to this aspect, the present invention provides a fusion protein comprising (i) at least one solubility-enhancing moiety of 100-160 amino acid residues having at least 70% identity with SEQ ID NO: 1 or SEQ ID NO: 96, wherein the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is selected from the group consisting of Asp and Glu; and (ii) at least one moiety which is a desired protein or polypeptide. Preferred features of the solubility-enhancing moiety are presented hereinabove. It has surprisingly been determined from experimental data that although these changes in positions 40 and 65 of the solubility-enhancing moiety do not alter the net charge of the resulting fusion protein, its capacity to provide stability and solubility to any desirable protein/polypeptide moiety to which it is fused is improved, and the solubility-enhancing moieties of the fusion protein are surprisingly prevented from dimerizing since electrostatic interactions involving the oppositely charged residues 40 and 65 are not formed.

In a preferred embodiment, the fusion proteins consists of (i) at least one solubility-enhancing moiety of 100-160 amino acid residues having at least 70% identity with SEQ ID NO: 1 or SEQ ID NO: 96, wherein the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is selected from the group consisting of Asp and Glu; and (ii) at least one moiety which is a desired protein or polypeptide, optionally including other preferred features disclosed herein, e.g. a linker peptide and/or a cleavage site between the solubility-enhancing moiety and the desired protein or polypeptide. In experiments, high yields of different fusion proteins have been achieved in E. coli. The fusion protein may be useful as such in isolated form, e.g. for studies of otherwise aggregated or poorly soluble proteins in soluble form, or in crystallization associated with X-ray crystallography. The fusion protein may also be cleaved to release the desired protein or polypeptide.

The term “fusion protein” implies here a protein that is made by expression from a recombinant nucleic acid, i.e. DNA or RNA that is created artificially by combining two or more nucleic acid sequences that would not normally occur together (genetic engineering). The fusion proteins according to the invention are recombinant proteins, and they are therefore not identical to naturally occurring proteins. The combined nucleic acid sequences encode different proteins, partial proteins or polypeptides with certain functional properties. The resulting fusion protein, or recombinant fusion protein, is a single protein with functional properties derived from each of the original proteins, partial proteins or polypeptides.

In certain embodiments, the fusion protein according to the invention and the corresponding genes are chimeric, i.e. the protein/gene fragments are derived from at least two different species. The solubility-enhancing moiety is derived from the N-terminal fragment of a spider silk protein. According to this aspect, it is preferred that the desired protein or polypeptide is a non-spidroin protein. This implies that the desired protein or polypeptide is not derived from the C-terminal, repetitive or N-terminal fragment of a spider silk protein. According to another aspect, it is preferred that the desired protein or polypeptide is a spidroin protein. This implies that the desired protein or polypeptide is derived from the C-terminal, repetitive or N-terminal fragment of a spider silk protein. The desired protein or polypeptide may also in turn be a fusion between a spidroin protein moiety and a non-spidroin polypeptide or protein moiety. Typically, the spidroin protein moiety provides the capacity of forming an ordered polymer, while the non-spidroin polypeptide or protein moiety may provide desirable affinity properties, e.g. cell-binding peptides, immunoglobulins and functional fragments thereof.

The fusion protein according to the invention may also contain one or more linker peptides. The linker peptide(s) may be arranged between the solubility-enhancing moiety and the desired protein or polypeptide moiety, or may be arranged at either end of the solubility-enhancing moiety and the desired protein or polypeptide moiety. If the fusion protein contains two or more solubility-enhancing moieties, the linker peptide(s) may also be arranged in between two solubility-enhancing moieties. The linker(s) may provide a spacer between the functional units of the fusion protein, but may also constitute a handle for identification and purification of the fusion protein, e.g. a His and/or a Trx tag. If the fusion protein contains two or more linker peptides for identification and purification of the fusion protein, it is preferred that they are separated by a spacer sequence, e.g. Hise-spacer-His6-. The linker may also constitute a signal peptide, such as a signal recognition particle substrate, which directs the fusion protein to the membrane and/or causes secretion of the fusion protein from the host cell into the surrounding medium. The fusion protein may also include a cleavage site in its amino acid sequence, which allows for cleavage and removal of the linker(s) and/or the solubility-enhancing moiety or moieties. Various cleavage sites are known to the person skilled in the art, e.g. cleavage sites for chemical agents, such as CNBr after Met residues and hydroxylamine between Asn-Gly residues, cleavage sites for proteases, such as thrombin or protease 3C, and self-splicing sequences, such as intein self-splicing sequences. A preferred cleavage site is after a Met residue.

Each solubility-enhancing moiety is linked, directly or indirectly, to the desired protein or polypeptide moiety. A direct linkage implies a direct covalent binding between the two moieties without intervening sequences, such as linkers. An indirect linkage also implies that the two moieties are linked by covalent bonds, but that there are intervening sequences, such as linkers and/or one or more further solubility-enhancing moieties.

The at least one solubility-enhancing moiety may be arranged at either end of the desired protein or polypeptide, i.e. C-terminally arranged or N-terminally arranged. It is preferred that the least one solubility-enhancing moiety is arranged at the N-terminal end of the desired protein or polypeptide. If the fusion protein contains one or more linker peptide(s) for identification and purification of the fusion protein, e.g. a His or Trx tag(s), it is preferred that it is arranged at the N-terminal end of the fusion protein. The at least one solubility-enhancing moiety may also be integrated within the desired protein or polypeptide, for instance between domains or parts of a desired protein. In a preferred embodiment, at least one solubility-enhancing moiety constitutes the N-terminal and/or the C-terminal end of the fusion protein, i.e. no linker peptide or other sequence is present terminal of the solubility-enhancing moiety. A typical fusion protein according to the invention may contain 1-6, such as 1-4, such as 1-2 solubility-enhancing moieties.

In a preferred embodiment, the fusion protein is comprising at least two solubility-enhancing moieties, each being derived from the N-terminal (NT) fragment of a spider silk protein as set out hereinabove. The solubility-enhancing moieties, preferably two solubility-enhancing moieties, may be consecutively arranged at either end of the desired protein or polypeptide, i.e. C-terminally arranged or N-terminally arranged. Consecutively arranged solubility-enhancing moieties may also be integrated within the desired protein or polypeptide, for instance between domains or parts of a desired protein. The solubility-enhancing moieties may also be non-consecutively arranged, either at each end of the desired protein or polypeptide, i.e. C-terminally and N-terminally arranged, or at one end of the desired protein or polypeptide and integrated within the desired protein or polypeptide. A typical preferred fusion protein according to the invention may contain 2-6, such as 2-4 solubility-enhancing moieties.

In a preferred embodiment, the fusion protein according to the invention has at least one cleavage site arranged between at least one desired protein or polypeptide moiety and at least one solubility-enhancing moiety. This allows for cleavage of the fusion protein and purification of the desired protein. It is however noted that it may be desirable to obtain the desired protein or polypeptide as part of a fusion protein, which may provide a suitable handle for purification and detection and/or provide desirable properties, e.g. stability and solubility. In this case, the cleavage site may be omitted, or the cleavage site may be included but the cleavage step omitted.

A preferred fusion protein has the form of an N-terminally arranged solubility-enhancing moiety, coupled by a linker peptide of 1-30 amino acid residues, such as 1-10 amino acid residues, to a C-terminally arranged desired protein or polypeptide. The linker peptide may contain a cleavage site. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.

Another preferred fusion protein has the form of an N-terminally arranged solubility-enhancing moiety coupled directly to a C-terminally arranged desired protein or polypeptide. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.

One preferred fusion protein has the form of a two consecutive N-terminally arranged solubility-enhancing moieties, coupled by a linker peptide of 1-30 amino acid residues, such as 1-10 amino acid residues, to a C-terminally arranged desired protein or polypeptide. The linker peptide may contain a cleavage site. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.

Another preferred fusion protein has the form of two consecutive N-terminally arranged solubility-enhancing moieties coupled directly to a C-terminally arranged desired protein or polypeptide. Optionally, the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.

In the context of the present invention, it is understood that by a desired polypeptide is meant a polypeptide of 5-50 amino acid residues, preferably 10-50, 20-50 or 40-50 amino acid residues. Further in the context of the present invention, it is understood that by a desired protein is meant a protein of more than 50 amino acid residues, such as more than 80 amino acid residues. It is preferred that the desired protein contains less than 500 amino acid residues, such as less than 300 or less than 200 amino acid residues. Preferred sizes of the desired polypeptide or protein are in the ranges of 4-50 kDa, such as 5-50 kDa, 4-45 kDa and 5-45 kDa, preferably 8-30 kDa, and more preferably 4-20, 5-20 or 8-30 kDa.

In preferred embodiments of the invention, the desired polypeptide or protein is hydrophobic, having an aliphatic index of 65 or higher, preferably 70 or higher, as determined by Protparam (web.expasy.org/protparam/; Gasteiger E. et al.; Protein Identification and Analysis Tools on the ExPASy Server; (in) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607). Preferably, the ratio of hydrophobic residues within the polypeptide, as defined by the Grand Average of Hydropathy (GRAVY) index (Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.) should be between −1 and 2, preferably between 1 and 2.

In one preferred embodiment, the desired protein or polypeptide is selected from the group consisting of amyloid-forming proteins and polypeptides, disulphide-containing proteins and polypeptides, apolipoproteins, membrane proteins and polypeptides, protein and polypeptide drugs and drug targets, aggregation-prone proteins and polypeptides, proteases, and immunoglobulins and fragments thereof. In a preferred embodiment, the desired protein or polypeptide is selected from the group consisting of amyloid-forming proteins and polypeptides, disulphide-containing proteins and polypeptides, apolipoproteins, membrane proteins and polypeptides, protein and polypeptide drugs and drug targets, aggregation-prone proteins and polypeptides, and proteases.

One preferred group of desired proteins or polypeptides is consisting of Aβ-peptide, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF, actin and β17; SP-B, mini-BLeu, α-defensins and β-defensins; class A-H apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, SP-C33Leu, KL4, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; SP-A, SP-D and analogues thereof; Bri2-BRICHOS and variants thereof, including Bri2₁₁₃₋₂₃₁; avidin and streptavidin; protease 3C; and immunoglobulins and fragments thereof. A preferred group of desired proteins or polypeptides is consisting of Aβ-peptide, IAPP, PrP, α-synuclein, calcitonin, prolactin, cystatin, ATF, actin and β17; SP-B, mini-BLeu, α-defensins and β-defensins; class A-H apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, SP-C33Leu, KL4, Brichos, GFP, eGFP, nicastrin, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; SP-A, SP-D and analogues thereof; Bri2-BRICHOS and variants thereof, including Bri2₁₁₃₋₂₃₁; avidin and streptavidin; and protease 3C.

Amyloid-forming proteins and polypeptides according to the invention include proteins and polypeptides that are associated with disease and functional amyloid. Examples of amyloid-forming proteins and polypeptides include amyloid beta peptide (Aβ-peptide), islet amyloid polypeptide (amylin or IAPP), prion protein (PrP), α-synuclein, calcitonin, prolactin, cystatin, atrial natriuretic factor (ATF) and actin. A further example is the designed polypeptide β17. Examples of amyloid-forming proteins and polypeptides according to the invention are listed in Table 2.

TABLE 2 Amyloid-forming proteins and polypeptides Uniprot ID/other Protein reference Aβ1-42 P05067 Apolipoprotein SAA P02735 Cystatin C P01034 Transthyretin P02766 Lysozyme P61626 α-synuclein P37840 Prion protein P04156 ODAM A1E959 Lactadherin Q08431 Tau P10636 Gelsolin P06396 ABri, ADan Q9Y287 Insulin P01308 Apolipoprotein A-II P02652 Apolipoprotein A-IV P06727 Semenogelin I P04279 Keratoepithelin Q15582 Lactotransferrin P02788 Fibrinogen α-chain P02671 ANF P01160 IAPP P10997 β2-microglobulin P61769 Calcitonin P01258 Prolactin P01236 Apolipoprotein A-I P02647 CsgA P28307 Sup35 C7GN25 Pmel17 P40967 HET-s A8HR89 Ure2p Q8NIE6 β17 SEQ ID NO: 27

Examples of disulphide-containing proteins and polypeptides include surfactant protein B (SP-B) and variants thereof, such as Mini-B, Mini-B27, Mini-BLeu, α-defensins and β-defensins. Without being limited to any specific theory, it is contemplated that the solubility-enhancing moiety promotes the desired formation of intrachain disulphide bonds over interchain disulphide bonds in defensins and other disulphide-containing proteins and polypeptides.

Examples of disulphide-containing proteins and polypeptides according to the invention are listed in Table 3.

TABLE 3 Disulphide-containing proteins and polypeptides Protein Sequence/Uniprot ID Human SP-B FPIPLPYCWLCRALIKRIQAMIPKGALAV (SEQ ID NO 28) AVAQVCRVVPLVAGGICQCLAERYSVILL DTLLGRMLPQLVCRLVLRCSM ^(a) Mouse SP-B LPIPLPFCWLCRTLIKRVQAVIPKGVLAV (SEQ ID NO 29) AVSQVCHVVPLVVGGICQCLAERYTVLLL DALLGRVVPQLVCGLVLRCST ^(a) Pig SP-B FPIPLPFCWLCRTLIKRIQAVVPKGVLLK (SEQ ID NO 30) AVAQVCHVVPLPVGGICQCLAERYIVICL NMLLDRTLPQLVCGLVLRCSS ^(a) Rabbit SP-B FPIPLPLCWLCRTLLKRIQAMIPKGVLAM (SEQ ID NO 31) AVAQVCHVVPLVVGGICQCLAERYTVILL EVLLGHVLPQLVCGLVLRCSS ^(a) Rat SP-B LPIPLPFCWLCRTLIKRVQAVIPKGVLAV (SEQ ID NO 32) AVSQVCHVVPLVVGGICQCLAERYTVLLL DALLGRVVPQLVCGLVLRCST ^(a) Mini-B CWLCRALIKRIQAMIPKGGRMLPQLVCRL (SEQ ID NO 33) VLRCS ^(b) Mini-BLeu CWLCRALIKRIQALIPKGGRLLPQLVCRL (SEQ ID NO 34) VLRCS ^(b) Mini-B27 CLLCRALIKRFNRYLTPQLVCRLVLRC ^(c) (SEQ ID NO 35) 1a AA CWLARALIKRIQALIPKGGRLLPQLVARL (SEQ ID NO 36) VLRCS ^(d) lb AA AWLCRALIKRIQALIPKGGRLLPQLVCRL (SEQ ID NO 37) VLRAS ^(e) 1a LL CWLLRALIKRIQALIPKGGRLLPQLVLRL (SEQ ID NO 38) VLRCS ^(d) lb LL LWLCRALIKRIQALIPKGGRLLPQLVCRL (SEQ ID NO 39) VLRLS ^(e) Proinsulin P01308 CAR D1 ^(f) P78310 ^(a) Cys8-Cys77, Cys11-Cys71, Cys35-Cys46 and intermolecular Cys48-Cys48 linkages ^(b) Cys1-Cys33 and Cys4-Cys27 linkages ^(c) Cys1-Cys27 and Cys4-Cys21 linkages ^(d) Cys1-Cys33 linkage ^(e) Cys4-Cys27 linkage ^(f) Coxsackievirus and adenovirus receptor

Examples of apolipoproteins include class A-H apolipoproteins. Examples of apolipoproteins according to the invention are listed in Table 4.

TABLE 4 Apolipoproteins Protein Sequence/Uniprot ID Apolipoprotein B-100 P04114 Apolipoprotein C-1 P02654 Apolipoprotein D P05090 Apolipoprotein E P02649

Examples of membrane proteins and polypeptides include membrane-associated receptors, including cytokine receptors, KL4, LL-37, hCAP18, surfactant protein C (SP-C) and variants thereof, such as SP-C (Leu), SP-C33, SP-C30 and SP-C33Leu. Other specific examples include SP-C33Leu fused to Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, la LL, 1b LL, 0 LLLL or SP-B proteins, optionally via a linker, e.g. Gly_(n), Leu_(n), Gly-Alan or the like. SP-C33Leu may be arranged N-terminal or, preferably, C-terminal to the Mini-B, Mini-BLeu, la AA, 1b AA, 0 AAAA, la LL, 1b LL, 0 LLLL or SP-B protein.

Examples of membrane proteins and polypeptides according to the invention are listed in Table 5.

TABLE 5 Membrane proteins and polypeptides SEQ Protein ID NO Sequence SP-C 40 FGIPCCPVHLKRLLIVVVVVVLIVVVI VGALLMGL * SP-C(Leu) 41 FGIPSSPVHLKRLKLLLLLLLLILLLI LGALLMGL SP-C33 42 IPSSPVHLKRLKLLLLLLLLILLLILG ALLMGL SP-C30 43 IPSSPVHLKRLKLLLLLLLLILLLILG ALL SP-C33(Leu) 44 IPSSPVHLKRLKLLLLLLLLILLLILG ALLLGL LL-37 45 LLGDFFRKSKEKIGKEFKRIVQRIKDF LRNLVPRTES KL4 46 KLLLLKLLLLKLLLLKLLLLK Uniprot Protein ID Growth hormone receptor P10912 G-protein coupled receptor 35 Q9HC97 Insulin receptor, P06213 Gonadotropin releasing hormone receptor P30968 Very low density lipoprotein receptor P98155 TGF-beta receptor, type 1 P36897 Prostaglandin D2 receptor Q13258 Receptor tyrosine-protein kinase P04626 erbB-2 (HER2) Receptor tyrosine-protein kinase Q15303 erbB-4 (HER4) Receptor tyrosine-protein kinase P21860 erbB-3 (HER3) Aquaporin-1 P29972 Aquaporin-2 P41181 Chloride channel protein CIC-Ka P51800 Chloride channel protein CIC-Kb P51801 Integral membrane protein DGCR2/IDD P98153 Interleukin 9 receptor Q01113 * Cys-5 and Cys-6 in native SP-C are palmitoylated

Examples of protein and polypeptide drugs and drug targets include hormones that are produced recombinantly, including peptide and protein hormones, such as erythropoietin (EPO) and growth hormone (GH), cytokines, growth factors, such as insulin-like growth factors (IGF-I and IGF-II), KL4, LL-37, hCAP18, surfactant protein C (SP-C) and variants thereof, such as SP-C (Leu), SP-C33, SP-C30, SP-C33Leu. Other specific examples include SP-C33Leu fused to Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL, 0 LLLL or SP-B proteins, optionally via a linker, e.g. Gly_(n), Leu_(n), Gly-Alan or the like. SP-C33Leu may be arranged N-terminal or, preferably, C-terminal to the Mini-B, Mini-BLeu, 1a AA, 1b AA, 0 AAAA, la LL, 1b LL, 0 LLLL or SP-B protein. Further preferred examples include surfactant proteins A (SP-A) and D (SP-D), and analogues thereof. A further preferred example is Bri2-BRICHOS and variants thereof, including Bri2₁₁₃₋₂₃₁, and nicastrin.

Examples of protein and polypeptide drugs and drug targets according to the invention are listed in Table 6.

TABLE 6 Protein and polypeptide drugs and drug targets Sequence/Uniprot ID/ Protein other reference Insulin-like growth factor IA P01243 Insulin like growth factor IB P05019 Growth hormone 1, variant 1 Q6IYF1 Growth hormone 1, variant 2 Q6IYF0 Growth hormone 2, variant 2 B1A4H7 Insulin P01308 Erythropoietin P01588 Coagulation Factor VIII P00451 Coagulation Factor IX P00740 Prothrombin P00734 Serum albumin P02768 Antithrombin III P01008 Interferon alfa P01563 Somatotropin P01241 Major pollen allergen P15494 Bet v 1-A OspA (Piscirickettsia Q5BMB7 salmonis) 17 kDa antigen variant of Q9F9K8 OspA (P. salmonis) Transforming growth factor P01137 beta-1 Transforming growth factor P61812 beta-2 Transforming growth factor P10600 beta-3 Interleukin 1 beta P01584 Interleukin 1 alfa P01583 Interleukin 2 P60568 Interleukin 3 P08700 Interleukin 4 P05112 Interleukin 5 P05113 Interleukin 6 P05231 Interleukin 7 P13232 Interleukin 8 P10145 Interleukin 9 P15248 Interleukin 10 P22301 Interleukin 12 subunit alfa P29459 Interleukin 12 subunit beta P29460 Interleukin 18 Q14116 Interleukin 21 Q9HBE4 Thymic stromal lymphopoietin Q969D9 hSP-A1 Q8IWL2 hSP-A1₈₁₋₂₂₈ SEQ ID NO 47: hSP-A2 Q8IWL1 hSP-A2₈₁₋₂₂₈ SEQ ID NO 48: hSP-D P35247 hSP-D₂₀₄₋₃₅₅ SEQ ID NO 49 Nicastrin Q92542 Bri2₁₁₃₋₂₃₁ (Bri2-Brichos) SEQ ID NO 50 Neuroserpin SEQ ID NO 51 SEQ ID Protein NO Sequence SP-C 40 FGIPCCPVHLKRLLIVVVVVVLIV VVIVGALLMGL ^(a) SP-C(Leu) 41 FGIPSSPVHLKRLKLLLLLLLLIL LLILGALLMGL SP-C33 42 IPSSPVHLKRLKLLLLLLLLILLL ILGALLMGL SP-C30 43 IPSSPVHLKRLKLLLLLLLLILLL ILGALL SP-C33(Leu) 44 IPSSPVHLKRLKLLLLLLLLILLL ILGALLLGL LL-37 45 LLGDFFRKSKEKIGKEFKRIVQRI KDFLRNLVPRTES KL4 46 KLLLLKLLLLKLLLLKLLLLK 1a AA 36 CWLARALIKRIQALIPKGGRLLPQ LVARLVLRCS ^(b) 1b AA 37 AWLCRALIKRIQALIPKGGRLLPQ LVCRLVLRAS ^(c) 0 AAAA 52 AWLARALIKRIQALIPKGGRLLPQ LVARLVLRAS 1a LL 38 CWLLRALIKRIQALIPKGGRLLPQ LVLRLVLRCS ^(b) 1b LL 39 LWLCRALIKRIQALIPKGGRLLPQ LVCRLVLRLS ^(c) 0 LLLL 53 LWLLRALIKRIQALIPKGGRLLPQ LVLRLVLRLS ^(a) Cys-5 and Cys-6 in native SP-C are palmitoylated ^(b) Cys1-Cys33 linkage ^(c) Cys4-Cys27 linkage

Examples of aggregation-prone proteins and polypeptides include avidin, streptavidin and extracellular, ligand-binding parts of cytokine receptors.

Examples of aggregation-prone proteins and polypeptides according to the invention are listed in Table 7.

TABLE 7 Aggregation-prone proteins and polypeptides Uniprot ID/other Protein reference Streptavidin, Streptomyces avidinii P22629 Streptavidin, Streptomyces lavendulae B8YQ01 Streptavidin V1, Streptomyces venezuelae Q53532 Streptavidin V2, Streptomyces venezuelae Q53533 Putative streptavidin, Burkholderia mallei A1V7Z0 (strain SAVP1) Putative streptavidin, Burkholderia Q2T1V4 thailandensis Putative streptavidin, Burkholderia mallei Q62EP2 Core streptavidin GenBank: CAA77107.1 M4 (quadruple mutein of streptavidin) J Biol Chem 280(24): 23225-23231 (2005) Avidin, Gallus gallus P02701 GenBank: CAC34569.1 Actin P68133 Interleukin 6 receptor subunit alfa P08887 Interleukin 6 receptor subunit beta P40189 Interleukin 2 receptor subunit alfa P01589 Interleukin 2 receptor subunit beta P14784 Cytokine receptor common subunit gamma P31785 Green Fluorescent Protein (GFP) SEQ ID NO 54 eGFP SEQ ID NO 55

Examples of proteases include protease 3C from coxsackie virus or human rhinovirus. Further examples of proteases according to the invention are listed in Table 8.

TABLE 8 Proteases Accession Protease Class no. Trypsin (bovine) serine P00760 Chymotrypsin (bovine) serine P00766 Elastase (porcine) serine P00772 Endoproteinase Arg-C (mouse submaxillary gland) serine Endoproteinase Glu-C (V8 protease) serine P04188 (Staphylococcus aureus) Acylamino-acid-releasing enzyme (porcine) serine P19205 Carboxypeptidase (Penicillium janthinellum) serine P43946 Proteinase K (Tritirachium album) serine P06873 Subtilisin (Bacillus subtilis) serine P04189 P29122 Carboxypeptidase Y (yeast) serine P00729 Endoproteinase Lys-C (Lysobacter enzymogenes) serine S77957 Enteropeptidase (human) serine P98073 Prothrombin serine P00734 Factor X serine P00742 Pepsin aspartic P00791 P00790 Cathepsin D (human) aspartic P07339 HIV-1 protease aspartic Q9YQ34 Cathepsin C cysteine Clostripain (endoproteinase-Arg-C) cysteine P09870 (Clostridium histolyticum) Papain (Carica papaya) cysteine P00784 Protease 3C cysteine Q04107 Tobacco Etch virus (TEV) cysteine Q0GDU8 Thermolysin (Bacillus thermo-proteolyticus) metallo P00800 Endoproteinase Asp-N (Pseudomonas fragi) metallo Q9R4J4 Carboxypeptidase A (bovine) metallo P00730 Carboxypeptidase B (porcine) metallo P00732 IgA protease metallo Q97QP7

In preferred embodiments of the invention, the desired protein or polypeptide is selected from surfactant protein B (SP-B) and variants thereof, such as Mini-B, Mini-B27, Mini-BLeu and KL4; Aβ, IAPP, β17; LL-37, hCAP18; surfactant protein C (SP-C) and variants thereof, such as SP-C (Leu), SP-C33, SP-C30 and SP-C33Leu; surfactant protein A (SP-A) and variants thereof; and surfactant protein D (SP-D) and variants thereof; and Bri2-BRICHOS and variants thereof, including Bri2₁₁₃₋₂₃₁. Other preferred proteins according to the invention are nicastrin, neuroserpin, GFP, eGFP, and the 1a AA, 1b AA, 0 AAAA, 1a LL, 1b LL and 0 LLLL proteins.

In certain preferred embodiments of the invention, the fusion protein is selected from the group consisting of SEQ ID NOS 56, 60, 64, 66, 70-75, 82, 84, 87, 90 and 93; and proteins having at least 80%, preferably at least 90%, more preferably at least 95% identity, to any of these proteins.

According to one aspect, the present invention provides a composition comprising an aqueous solution of a protein according to the invention. In a preferred embodiment, the composition is consisting of an aqueous solution of a protein according to the invention. It is preferred that the protein is a fusion protein according to the invention. It is preferred that the pH of the composition is 7.2 or lower, such as 5.5-7.2.

According to another aspect, the present invention provides an isolated nucleic acid, preferably a cDNA, encoding a protein according to the invention. In a preferred embodiment, the isolated nucleic acid is selected from the group consisting of SEQ ID NOS 58, 62, 65, 68, 76-81, 83, 85, 89, 91 and 95.

According to one aspect, the present invention provides a novel use of at least one moiety of 100-160 amino acid residues having at least 70% identity with SEQ ID NO: 1, wherein the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is selected from the group consisting of Asp and Glu, as a moiety in a fusion protein for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide as set out herein. Preferred features of the inventive solubility-enhancing moiety are presented hereinabove.

In one preferred embodiment, the solubility-enhancing moiety is used for production of the desired protein or polypeptide. In another preferred embodiment, the solubility-enhancing moiety is used for studying or characterizing the desired protein or polypeptide.

An advantageous use of the inventive moiety is as a solubility-enhancing moiety in a fusion protein which is subjected to a pH of 7.2 or lower, such as 5.5-7.2. This specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wildtype NT forms dimers at a pH interval of 5.5-7.2 which increases the risk of undesirable aggregation of the fusion proteins. This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide. It is also a useful pH interval for certain purification protocols, e.g. when using ion exchange, such as cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) as a purification principle. It is also a useful pH interval for certain expression hosts, e.g. yeasts.

According to another aspect, the present invention provides a method of producing a desired protein or polypeptide. The first step involves expressing in a suitable host a fusion protein according to the invention, comprising the desired protein or polypeptide. The solubility-enhancing moiety has at least 70% identity, such as 75%, 80%, 85%, 90%, 95% or even 100% identity with any one of the charge-reversed NT proteins as set out in SEQ ID NO: 1-7 or the charge-reversed variants of SEQ ID NO: 96, preferably SEQ ID NO: 1-2 or the charge-reversed variants of SEQ ID NO: 96. As set out herein, the amino acid residue corresponding to position 40 in SEQ ID NO: 1 is selected from the group consisting of Lys, Arg and His; and the amino acid residue corresponding to position 65 in SEQ ID NO: 1 is selected from the group consisting of Asp and Glu. In an alternative embodiment, the solubility-enhancing moiety has at least 70% identity, such as 75%, 80%, 85%, 90%, 95% or even 100% identity with the wildtype NT protein as set out in SEQ ID NO: 9 or 96. For avoidance of doubt, this implies that the method is also applicable for solubility-enhancing moieties which are not charge-reversed NT proteins, e.g. not as set out in SEQ ID NO: 1-7.

Suitable expression hosts are well known to a person skilled in the art and include e.g. bacteria and eukaryotic cells, such as yeast, insect cell lines and mammalian cell lines. Typically, this step involves expression of a nucleic acid molecule which encodes the fusion protein in E. coli.

The second method step involves obtaining a mixture containing the fusion protein, and optionally isolating the fusion protein. The mixture may for instance be obtained by lysing or mechanically disrupting the host cells. The mixture may also be obtained by collecting the cell culture medium, if the fusion protein is secreted by the host cell. The thus obtained protein can be isolated using standard procedures. If desired, this mixture can be subjected to centrifugation, and the appropriate fraction (precipitate or supernatant) be collected. The mixture containing the fusion protein can also be subjected to gel filtration, chromatography, e.g. ion exchange chromatography, such as cation or anion exchange chromatography, dialysis, phase separation or filtration to cause separation.

In a preferred embodiment, the obtained mixture comprises the fusion protein dissolved in a liquid medium, typically a salt buffer or cell culture medium. This specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wildtype NT forms dimers at a pH interval of 5.5-7.2 which increases the risk of undesirable aggregation of the fusion proteins. This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide, e.g. amyloid-forming or aggregation-prone proteins/polypeptides. It is also a useful pH interval for certain expression hosts, e.g. yeasts. It is also a useful pH interval for certain purification protocols, e.g. when using ion exchange, such as cation or anion exchange, or immobilized metal ion affinity chromatography (IMAC) as a purification principle. In a preferred embodiment, this step further involves purification of the fusion protein on an ion exchange medium, such as a cation or anion exchange medium. In one preferred embodiment, this step further involves purification of the fusion protein on an IMAC medium, preferably with elution using low pH (below the pKa of His, typically a pH of approximately 6).

Thus, the fusion protein is typically obtained as a solution in a liquid medium. By the terms “soluble” and “in solution” is meant that the fusion protein is not visibly aggregated and does not precipitate from the solvent at 60 000×g. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as a 10-50 mM Tris-HCl buffer or phosphate buffer.

It has been advantageously been found that the presence of the solubility-enhancing moiety according to the invention improves the stability of the desired protein/polypeptide and prevents moiety dimer formation under these conditions. This can be advantageous when immediate polymerisation may be undesirable, e.g. during protein purification or in preparation of large batches. In particular, this is advantageous for methods according to the invention which are comprising at least one step involves subjecting the fusion protein to a pH of 7.2 or lower, such as 5.5.-7.2. As set out above, this specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wildtype NT forms dimers at a pH interval of 5.5-7.2 which increases the risk of undesirable aggregation of the fusion proteins. This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide, e.g. amyloid-forming or aggregation-prone proteins/polypeptides. It is also a useful pH interval for certain expression hosts, e.g. yeasts.

In a preferred embodiment, the fusion protein is isolated from the mixture without any separation step involving gel filtration, chromatography or any other solid phase adsorption-based separation. In one preferred embodiment, the fusion protein is isolated from the mixture by precipitation of the fusion protein, followed by suspending the precipitated fusion protein in an aqueous solvent, wherein the fusion protein is soluble in the aqueous solvent.

The precipitation of the fusion protein may be performed by any suitable technique to decrease the solubility of the fusion protein in the solvent, including changes in ionic strength and addition of miscible organic solvents. It is preferred that the precipitation of the fusion protein is achieved by salting out at high salt concentration, i.e. subjecting the fusion protein to sufficiently high salt concentration to render the fusion protein insoluble. By way of example, a NaCl concentration of 0.5 M or higher, such as 1 M or higher, is typically sufficient to precipitate the fusion protein. The precipitate containing the fusion protein is collected e.g. by filtration or centrifugation, and the filtrate or supernatant is discarded. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, the isolated precipitated fusion protein can be suspended and dissolved in a suitable solvent. Preferably, the precipitated fusion protein is suspended in an aqueous solvent, wherein the fusion protein is soluble.

In certain embodiments, the method is further comprising the step of cleaving the fusion protein to release the desired protein or polypeptide from the residual solubility-enhancing moiety or fragments thereof as cleavage products; and optionally isolating the desired protein or polypeptide. If desired, linker peptides may be removed by cleavage in this step. In these embodiments, the fusion protein is comprising at least one cleavage site arranged between at least one desired protein or polypeptide moiety and at least one solubility-enhancing moiety. In a typical fusion protein, this implies the presence of a single cleavage site between the solubility-enhancing moiety or moieties and the desired protein or polypeptide. Cleavage may be achieved using standard procedures, for instance cleavage by cyanogen bromide (CNBr) after Met residues, cleavage by hydroxylamine between Asn and Gly residues, cleavage by protease 3C between Gln and Gly residues at -XLETLFQGX- sites, and at various other protease sites that are well known to the person skilled in the art.

The thus obtained desired protein or polypeptide can be isolated using standard procedures. If desired, this mixture can also be subjected to centrifugation, and the appropriate fraction (precipitate or supernatant) be collected. The mixture containing the desired protein or polypeptide can also be subjected to gel filtration, chromatography, dialysis, phase separation or filtration to cause separation. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, linker peptides may be removed by cleavage in this step.

In a preferred embodiment, the desired protein or polypeptide is isolated from the mixture without any separation step involving gel filtration, chromatography or any other solid phase adsorption-based separation. In one preferred embodiment, the desired protein or polypeptide is isolated from the mixture by extraction in an organic solvent, i.e. extracting the desired protein or polypeptide by suspending the cleavage products in an organic solvent wherein the desired protein or polypeptide is soluble and wherein the residual solubility-enhancing moiety according to the invention or fragments thereof is/are not soluble.

The organic solvents that are used according to the invention are carbon-containing solvents and may exhibit a varying degree of polarity. Although termed “solvents”, it shall be understood that these organic solvents are utilized for balancing and shifting the solubility of the fusion protein and/or the desired protein or polypeptide and/or the residual solubility-enhancing moiety or fragments thereof during the manufacturing method. The proteins or polypeptides may very well be dissolved in an organic solvent at a certain organic solvent concentration interval, but falls out and forms a precipitate when the organic solvent concentration is increased or decreased. For instance, the residual solubility-enhancing moiety or fragments thereof can be dissolved in a 50/50 (vol/vol) mixture of an organic solvent, e.g. a lower alkyl alcohol, and water, but falls out and forms a precipitate in 90/10 or 10/90 (vol/vol) mixtures. When subjected to non-precipitating conditions, e.g. a 50/50 or a 0/100 mixture, the proteins or polypeptides return to the non-precipitated, dissolved state. The skilled person is well aware that other factors may have an impact on the limiting organic solvent(s) concentration for precipitation of the proteins or polypeptides, such as temperature, pH, ion strength and type of organic solvent(s). The limiting concentration for precipitation of the proteins or polypeptides under given conditions is well known or can easily be determined by a skilled person in the field.

Without being limited thereto, the organic solvents according to the invention can be selected from the group consisting of pentane, hexane, cyclohexane, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, chloroform, ethyl acetate, acetamide, diethyl ether, tetrahydrofurane, acetonitrile, methyl ethyl ketone, acetone, lower alkyl alcohols, e.g. methanol, ethanol, propanol, isopropanol and butanol, or any mixture of the mentioned solvents. It is preferable that the organic solvents according to the invention are water-soluble. A preferred group of organic solvents is the lower alkyl alcohols. The term lower alkyl alcohol includes primary, secondary and tertiary alkyl alcohols having from one to six carbon atoms, i.e. C1-6 alkyl alcohols. Specific examples of lower alkyl alcohols include methanol, ethanol, denatured spirit, n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Preferred lower alkyl alcohols are methanol, ethanol, isopropanol and isobutanol, in particular ethanol, due to price, availability and easy handling.

In one preferred embodiment, the method is further comprising, prior to the extraction step, the step of precipitation of the cleavage products. The precipitation of the cleavage products may be performed by any suitable technique to decrease the solubility of the desired protein or polypeptide in the solvent, including changes in ionic strength and addition of miscible organic solvents. It is preferred that the precipitation of the cleavage products is achieved by salting out at high salt concentration, i.e. subjecting the cleavage products to sufficiently high salt concentration to render the desired protein or polypeptide insoluble. By way of example, a NaCl concentration of 0.5 M or higher, such as 1 M or higher, is typically sufficient to precipitate the desired protein or polypeptide. The precipitate containing the desired protein or polypeptide is collected e.g. by filtration or centrifugation, and the filtrate or supernatant is discarded. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, the isolated precipitated desired protein or polypeptide can be suspended and dissolved in a suitable solvent.

A preferred method of producing a desired protein or polypeptide is thus comprising the following steps:

a) expressing in a suitable host a fusion protein according to the invention, comprising the desired protein or polypeptide; and b) obtaining a mixture containing the fusion protein, and

b1) isolating the fusion protein, comprising the following steps:

-   -   b1a) precipitation of the fusion protein, preferably by salting         out at high salt concentration; and     -   b1b) suspending the precipitated fusion protein in an aqueous         solvent, wherein the fusion protein is soluble in the aqueous         solvent;         c) cleaving the fusion protein to release the desired protein or         polypeptide from the residual solubility-enhancing moiety or         fragments thereof as cleavage products; and

c1) isolating the desired protein or polypeptide, comprising the following steps:

-   -   c1a) precipitation of the cleavage products; preferably by         salting out at high salt concentration;     -   c1b) extracting the desired protein or polypeptide by suspending         the precipitated cleavage products in an organic solvent,         preferably comprising a lower alkyl alcohol, such as methanol,         ethanol or isopropanol; wherein the desired protein or         polypeptide is soluble in the organic solvent; and wherein the         residual solubility-enhancing moiety or fragments thereof is/are         not soluble in the organic solvent.

The present invention will in the following be further illustrated by the following non-limiting examples.

EXAMPLES Example 1—Expression of NT and Charge-Reversed NT Mutants

Constructs with NT_(wt) (SEQ ID NO: 12, encoding SEQ ID NO: 11) and NT_(D40K/K65D) (SEQ ID NO: 8, encoding SEQ ID NO: 2) were cloned into pT7 expression vectors and transformed into chemically competent E. coli BL21 (DE3) cells. Plasmid-containing cells were inoculated to 10 mL Luria-Bertani (LB) medium with 70 mg/L kanamycin and grown at 37° C. and 180 rpm over night. 5 mL over-night culture was inoculated to 500 mL LB medium (1/100) with kanamycin and cells were further grown at 30° C. to OD₆₀₀ of ^(˜)1. The cells were induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and expression was performed at 20° C. over night. The day after, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8 to 30 mL and stored at −20° C. for at least 24 hours.

Example 2—Biophysical Properties of NT_(D40K/K65D) Compared to NT_(wt)

The NT dimerization process is highly dependent on intermolecular electrostatic interactions between the residues D40 and K65, playing a key role in the initial association of monomers. In this study, we designed and evaluated a double mutant (NT_(D40K/K65D); SEQ ID NO: 2) where these residues were swapped compared to the wildtype NT (NT_(wt); SEQ ID NO: 11), while preserving the net charge of the domain. Important biophysical properties were evaluated to determine the applicability of the mutant as a solubility enhancing fusion partner.

(A) Tryptophan Fluorescence Measurement

Fluorescence emission spectra for NT_(D40K/K65D) (SEQ ID NO: 2) and NT_(wt) (SEQ ID NO: 11), respectively, were measured on a spectrofluorometer (Tecan Safire 2) using Costar® black polystyrene assay plates with 96 flat bottom wells. The proteins were diluted to a concentration of 5 μM in 20 mM HEPES/20 mM MES adjusted to pH 5.6-8.0 in steps of 0.4 pH units. After exciting the samples at 280 nm (5 nm bandwidth), emission spectra were recorded in 1 nm steps between 300-400 nm (10 nm bandwidth). The tryptophan fluorescence ratio was calculated from the intensities at 339 nm and 351 nm and plotted as a function of pH. The data obtained for NT_(wt), was fitted to a two-state binding model due to the sigmoidal behavior of the monomer-dimer equilibrium.

The pH-dependent monomer-dimer equilibrium of NT can be monitored through the fluorescence shift of a single tryptophan (Trp) residue that becomes more exposed in the dimer. FIG. 2 is a graph illustrating the monomer-dimer equilibrium measured with Trp fluorescence. Trp fluorescence spectra between 300 and 400 nm were measured in 20 mM HEPES/20 mM MES buffer and the ratio at 339/351 nm (wavelengths corresponding to monomer and dimer conformations, respectively) was calculated and plotted as a function of pH for NT_(wt) (dotted line) and NT_(D40K/K65D) (filled line). The ratio of fluorescence at 339 and 351 nm as a function of pH gives a sigmoidal plot for NT_(wt) with a pKa of dimerization at pH 6.5. This fluorescence shift was not observed for the mutant NT_(D40K/K65D) and a ratio corresponding to a monomer was measured over the whole pH range (FIG. 2).

(B) [¹⁵N, ¹H]-HSQC NMR Measurements

Cells expressing the NT variants NT_(D40K/K65D) (SEQ ID NO: 2) and NT_(wt) (SEQ ID NO: 11) were cultured over-night and further inoculated 1/100 to 500 mL minimal medium M9 containing ¹⁵N-labelled ammonium chloride and 70 mg/L kanamycin. The cells were grown over night at 22° C. to an OD₆₀₀ of 1.4. Protein was expressed and purified as previously described.

[¹⁵N, ¹H]-HSQC NMR spectra were acquired at 25° C. on a Varian Unity Inova 600-MHz NMR spectrometer equipped with an HCN cold probe. NMR samples of ¹⁵N-labelled protein were prepared in either 20 mM sodium phosphate, 20 mM NaCl, pH 5.5 or 20 mM sodium phosphate, 300 mM NaCl, pH 7.2 buffers and 2D [¹⁵N, ¹H]-HSQC NMR spectra were recorded. The spectra were processed and analyzed using Bruker Topspin 3.1 software.

HSQC NMR was measured at pH 7.2 and 5.5 where NT_(wt) is a monomer or dimer, respectively, showing large chemical shift differences. Overlays of ¹⁵N-¹H HSQC NMR spectra of NT_(wt) and NT_(D40K/K65D) were produced at pH 5.5 and at pH 7.2 (not shown). FIG. 3 shows averaged backbone amide 1H and 15N chemical shift differences Δδ_(av)=√{square root over ((0.1Δδ_(N))²+(Δδ_(H))²)} between NT_(wt) and NT_(D40K/K65D) at pH 5.5 (lower panel) and pH 7.2 (upper panel). The spectra obtained from NT_(D40K/K65D) were similar at both pH values and corresponding to the spectra for monomeric NT_(wt) at pH 7.2.

(C) Urea-Induced Denaturation as a Measure of Stability

NT_(wt) (SEQ ID NO: 11) and NT_(D40K/K65D) (SEQ ID NO: 2) proteins were diluted to 5 μM in 20 mM HEPES/20 mM MES supplemented with 0-7 M urea in 0.5 M steps. The stability of the proteins at each concentration of urea was monitored with Trp fluorescence at constant pH values ranging from 5.0 to 7.5 with 0.5 unit steps. For each measured pH, the fluorescence ratio was plotted against the urea concentration and fitted to a two-state unfolding model in order to determine the transition points. The data for NT_(wt) (dotted line) and NT_(D40K/K65D) (filled line) are presented as transition points between native and denatured states ([den]^(50%)) as a function of pH in FIG. 4.

Referring to FIG. 4, NT_(wt) is significantly more stable in the dimer conformation at low pH. In contrast, NT_(D40K/K65D) exhibits an increased overall stability in the entire pH interval, similar to the NT_(wt) dimer and independent of changes in pH.

(D) Temperature Scans as a Measure of Stability

Temperature scans were performed and analyzed with circular dichroism (CD). Experiments were performed on a 410-model circular CD spectrometer (Aviv biomedical Inc., Lakewood, N.J., USA) using 300 μL cuvettes with a 1 mm path length. For all measurements, the NT_(wt) (SEQ ID NO: 11) and NT_(D40K/K65D) (SEQ ID NO: 2) proteins were diluted to 10 μM in 5 mM phosphate buffer at pH 5.5 or pH 8.0. CD spectra were recorded from 260 to 185 nm at 25° C., after heating to 95° C. and again at 25° C. after the samples were allowed to cool down. For each temperature, the data is shown as an average of 4 scans. Temperature scans were measured at 222 nm by recording 1° C. steps in the temperature interval 25-95° C., and data were fitted to a two-state unfolding model.

FIG. 5 shows thermal stability of NT_(wt) and NT_(D40K/K65D) measured with CD spectroscopy. The CD signal in mdegrees was measured at 222 nm at pH 5.5 and pH 8.0, plotted as a function of temperature (° C.) and fitted to a two-state unfolding model to obtain the melting temperatures (T_(m)) at the equilibration points. The highest melting temperatures were determined for NT_(D40K/K65D) at pH 8.0 and pH 5.5, respectively. Considerably lower melting temperatures were determined for NT_(wt) monomer at pH 8.0 and the NT_(wt) dimer at pH 5.5.

FIG. 6 shows refolding capacity of NT_(wt) and NT_(D40K/K65D) measured with CD spectroscopy. The molar ellipticity was scanned between 185 nm and 260 nm at 25° C., 95° C., and again at 25° C. after cooling for NT_(wt) at (A) pH 8.0 and (B) pH 5.5 and for NT_(D40K/K65D) at (C) pH 8.0 and (D) pH 5.5. The data is presented as a smoothed average of four measurements. CD spectra monitored before and after thermal denaturation shows that although NT_(wt) refolds into an alpha-helical structure, there is a reduced ellipticity after refolding at pH 8 (FIG. 6A), which is even more evident at pH 5.5 (FIG. 6B). An increased refolding capacity was observed for NT_(D40K/K65D), showing close to identical alpha-helical spectra before temperature induced unfolding and after refolding at pH 8 (FIG. 6C) and pH 5.5 (FIG. 6D).

Example 3—Expression of Fusion Proteins

The fusion protein constructs with the target peptides SP-C33Leu (SEQ ID NO: 58-59, encoding SEQ ID NO: 56-57) and KL4 (SEQ ID NO: 62-63, encoding SEQ ID NO: 60-61) in fusion with NT_(D40K/K65D) and NT_(wt), respectively, were cloned into pT7 expression vectors and transformed into chemically competent E. coli BL21 (DE3) cells. Fusion protein constructs with the same target peptides and proteins in fusion with PGB1 or Trx were subjected to the same procedure.

Plasmid-containing cells were inoculated to 10 mL LB medium with 70 mg/L kanamycin and grown at 37° C. and 180 rpm over night. 5 mL over-night culture was inoculated to 500 mL LB medium (1/100) with kanamycin and cells were further grown at 30° C. to ° Dm) of ^(˜)1. The cells were induced by addition of IPTG to a final concentration of 0.5 mM and expression was performed at 20° C. over night. The day after, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8 to 30 mL and stored at −20° C. for at least 24 hours.

Example 4—Purification of Fusion Proteins for Comparison of Yields

The ability of the NT variants NT_(wt) and NT_(D40K/K65D) to mediate solubility to aggregation-prone fusion partners in comparison to the highly soluble PGB1 domain was tested. Trx was also evaluated in fusion with SP-C33Leu but was later excluded due to its poor performance.

Fusion proteins obtained in Example 3 were solubilized by sonication in loading buffer (20 mM Tris-HCl, pH 8) at 80% amplitude, 1 s on and 1 s off for a total of 3 min. The soluble and insoluble fractions were separated by centrifugation at 27 000×g, 4° C. for 30 min. The clear lysate was loaded to an IMAC column, previously packed with Ni-Sepharose (GE Healthcare) and equilibrated with loading buffer. Bound protein was washed with 20 mM Tris-HCl, 5 mM imidazole, pH 8 and eluted with 20 mM Tris-HCl, 300 mM imidazole, pH 8 in 1 mL fractions. The absorbance at 280 nm was measured for each fraction, and protein-rich fractions were pooled. Imidazole was removed by over-night dialysis at 4° C. and in 5 L loading buffer, using a Spectra/Por® dialysis membrane with a 6-8 kDa molecular weight cut-off. The purity of the protein in each step was determined by SDS-PAGE using a 15% acrylamide gel stained with Coomassie Brilliant Blue.

(A) SP-C33Leu Fusion Proteins

FIG. 7 shows an SDS-PAGE evaluation of SP-C33Leu fusion proteins. The peptide was fused C-terminally of NT_(wt) (lane 1), NT_(D40K/K65D) (lane 2), Trx (lane 3) or PGB1 (lane 4). Lane M denotes the size marker and the molecular weights are indicated to the left. Panel A shows an expression analysis before induction and after over-night expression at 20° C. in Bl21 E. coli cells. Panel B shows a solubility analysis after 3 min of sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. Panel C shows fusion proteins after comparative Ni-Sepharose purification.

(B) KL4 Fusion Proteins

FIG. 8 shows an SDS-PAGE evaluation of KL4 fusion proteins. The peptide was fused C-terminally of NT_(wt) (lane 1), NT_(D40K/K65D) (lane 2) or PGB1 (lane 3). Lane M denotes the size marker and the molecular weights are indicated to the left. Panel A shows an expression analysis before induction and after over-night expression at 20° C. in Bl21 E. coli cells. Panel B shows a solubility analysis after 3 min of sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. Panel C shows fusion proteins after comparative Ni-Sepharose purification.

In summary, the NT variants NT_(wt), and NT_(D40K/K65D) were abundantly expressed at similar levels in fusion with SP-C33Leu (FIG. 7A) and KL4 (FIG. 8A), and at higher levels compared to PGB1 and Trx. All proteins and peptides appeared stable in fusion with the NT variants NT_(wt), and NT_(D40K/K65D), but Trx-SP-C33Leu showed signs of degradation during expression.

Both NT_(D40K/K65D) and PGB1 were able to mediate high solubility to their target proteins/peptides and with some minor differences, the most part was found in the soluble fractions (FIG. 7-8B). NT_(wt) showed ˜50% soluble protein in fusion with KL4 (FIG. 8B). The most remarkable difference between the NT variants was observed in fusion with SP-C33Leu, resulting in mainly insoluble protein for NT_(wt) and fully soluble protein for NT_(D40K/K65D) (FIG. 7B).

Purification of NT_(D40K/K65D) fusion proteins on Ni-sepharose yielded 284 and 428 mg/L culture for SP-C33Leu and KL4, respectively (FIG. 7-8C). This corresponds to between 2 and 8-fold higher amounts compared to protein in fusion with PGB1, mainly due to the higher expression levels (Table 9). The yields for NT_(wt) were intermediate, around 1.3 to 4-fold higher compared to PGB1 fusions proteins. Trx in fusion with SP-C33Leu gave the lowest yield and the fusion protein continued to degrade during purification (FIG. 7C).

TABLE 9 Calculated yields after Ni-Sepharose purification of fusion proteins Purified fusion protein (mg/L culture) Target for solubility-enhancing moiety protein/peptide NT_(wt) NT_(D40K/K65D) PGB1 Trx SP-C33Leu 93 284 56 43 KL4 212 428 56 —

Example 5—Purification of SP-C33Leu and KL4 Peptides

Cells expressing fusion proteins obtained in Example 3 were lysed by sonication at 80% amplitude for 1.5 min, 1 s on and 1 s off, 3 min total time. Only during full-scale purification by precipitation, the sonication procedure was repeated after standing on ice for 5 min and the sample was centrifuged at 50 000×g for 30 min. Sodium chloride was added to the supernatant to a final concentration of 1.2 M and the centrifugation was repeated. The pellet from the centrifugation was dissolved in 20 mM Tris-HCl, pH 8 and briefly sonicated at 60% amplitude for 1.5 min, 1 s on and 1 s off, 3 minutes in order to fully re-dissolve the fusion protein. CNBr cleavage was performed by adding 1.7 mL 2 M HCl to 30 mL dissolved solution, followed by 1.7 mL 1 M CNBr. The cleavage reaction was performed over night at room temperature. The next day, 800 mM sodium chloride was added to the cleavage reaction in a second precipitation step, followed by centrifugation at 20 000×g for 30 min. The supernatant was removed and the pellet was dried at 37° C. and suspended in 99.9% ethanol. Insoluble material was removed by centrifugation at 20 000×g for 30 min.

(A) SP-C33Leu Peptide (SEQ ID NO: 44)

SP-C33Leu was expressed as a NT_(D40K/K65D) fusion protein and produced in a process independent of chromatographic steps. FIG. 7D shows an SDS-PAGE evaluation of purification of the SP-C33Leu peptide from NT_(D40K/K65D)-SP-C33Leu using a NaCl precipitation/ethanol extraction protocol and CNBr bromide cleavage for removal of the fusion tag. P1, S1, P2, S2 and T denote insoluble fraction, soluble fraction, pellet after first precipitation, supernatant after first precipitation, and purified target peptide, respectively.

First, the sonicated cell lysate was purified in one simple step using 1.2 M sodium chloride to precipitate the majority of the fusion protein and remove most contaminants (FIG. 7D). The fusion protein was designed with a methionine residue located just N-terminally of the peptide, allowing for cleavage with cyanogen bromide (CNBr).

Subsequent to CNBr cleavage under acidic conditions, a second precipitation was performed using 0.8 M sodium chloride. SP-C33Leu and KL4 are both soluble in organic solvents, e.g. ethanol, methanol or isopropanol, and surprisingly all the NT-fragments generated by CNBr remain insoluble in these solvents. Accordingly, the precipitated pellet was further purified by suspension in 99.9% ethanol followed by centrifugation to isolate 20-30 mg/L culture of highly pure SP-C33Leu peptide in the soluble ethanol fraction (FIG. 7D).

(B) KL4 Peptide (SEQ ID NO: 46)

The procedure set out above for purification of the SP-C33Leu peptide from NT_(D40K/K65D)-SP-C33Leu was reproducible also for purification of the KL4 peptide from NT_(D40K/K65D)-KL4. FIG. 8D shows an SDS-PAGE evaluation of purification of the KL4 peptide from NT_(D40/K65D)-KL4 using a NaCl precipitation/ethanol extraction protocol and CNBr bromide cleavage for removal of the fusion tag. P1, S1, P2, S2 and T denote insoluble fraction, soluble fraction, pellet after first precipitation, supernatant after first precipitation, and purified target peptide, respectively.

Ethanol extraction yielded 10-15 mg/L culture of pure KL4 peptide (FIG. 8D).

Example 6—ESI-MS Characterization of SP-C33Leu

Further characterization of the purified SP-C33Leu (SEQ ID NO: 44) obtained in Example 5 with ESI-MS showed that the recombinantly produced peptide dissolved in ethanol has the correct covalent structure.

ESI-MS spectra of SP-C33Leu obtained in Example 5 are shown in FIG. 9. The spectrum shown in panel A shows mainly monomeric SP-C33Leu with 3 or 4 charges and one or two sodium adducts, and a minor fraction of dimers with 7 charges. A small amount of a contaminant (approximately 4314.8 Da corresponding to the peak at 1079 m/z) could also be observed. Panel B shows a MS/MS spectrum of the m/z 1199.2 peak in panel A.

Example 7—Effect of rSP-C33Leu on Tidal Volumes and Lung Gas Volumes

The effect of SP-C and derivatives thereof on tidal volumes and lung gas volumes can be evaluated using an animal model with positive end-expiratory pressure (PEEP) (Almlen, A et al., Neonatology 92, 194-200 (2007)).

Immature newborn rabbits (gestational age 27 days) were treated at birth with 200 mg/kg of 2% rSP-C33Leu (SEQ ID NO: 44) in dipalmitoylphosphatidylcholine (DPPC)/palmitoyloleoyl-phosphatidylglycerol (POPG) 68:31 (w/w) at a concentration of 80 mg/ml. Animals receiving the same dose of Curosurf® served as positive and non-treated littermates as negative controls. Animals were ventilated with a standard pressure sequence for 35/0 (peak-insufflation pressure [cm H₂O]/positive end-expiration pressure (PEEP) [cm H₂O]) for 1 min, 23/3 for 15 min, 18/3 for 5 min, 13/3 for 5 min and 23/3 for 5 min. Finally, the lungs were ventilated for additional 5 min with nitrogen at 23/3 cm H₂O and then excised for gas volume measurements. Both tidal volumes and lung gas volumes are given as median values.

FIG. 10 illustrates the effects of rSP-C33Leu on tidal volumes. Tidal volumes during 30 min of ventilation are shown for immature newborn rabbits treated at birth with 200 mg/kg of 2% rSP-C33Leu in DPPC:POPG (68:31 w/w) at a concentration of 80 mg/mL and compared to animals receiving the same dose of Curosurf, DPPC:POPG only (negative control) and non-treated animals. The tidal volumes are markedly increased for animals treated with 2% rSP-C33Leu in DPPC:POPG (68:31), compared to untreated negative controls and controls treated with DPPC:POPG (68:31), and approach those obtained after treatment with Curosurf®.

FIG. 11 illustrates the effects of rSP-C33Leu on lung gas volumes. Lung gas volumes are shown for immature newborn rabbits treated at birth with 200 mg/kg of 2% rSP-C33Leu in DPPC:POPG (68:31 w/w) at a concentration of 80 mg/mL and compared to animals receiving the same dose of Curosurf®, DPPC:POPG only (negative control) and non-treated animals. The lung gas volumes of animals treated with 2% rSP-C33Leu in DPPC:POPG (68:31) and Curosurf are equal, and significantly higher than those for animals treated with DPPC:POPG (68:31). Similar results were obtained using a synthetic SP-C33Leu peptide in the same animal model (data not shown).

Example 8—Expression of β17 Polypeptide Using a NT_(D40K/K65D) Fusion Protein

In order to investigate amyloid aggregation mechanisms broadly, researchers have designed β17 (SEQ ID NO: 27), a polypeptide that is very aggregation-prone and able to form amyloid-like fibrils in vitro. β17 is composed of 6 β-strands containing 7 amino acids each, separated by 5 short turns. The residues are disposed in a polar non-polar pattern. β17 has previously been expressed together with a myc-tag for immunodetection, and consequently, the peptide was not soluble and had to be purified from inclusion bodies. Such a purification process requires the use of denaturing conditions (8 M urea), is time consuming and yields an unstable protein. Moreover, studies of β17 fibril formation using Thioflavin T (ThT) as a reporter has been troubled with precocious aggregation.

Constructs with β17 polypeptide in fusion with NT_(D40K/K65D) (SEQ ID NO: 65, encoding SEQ ID NO: 64) and PGB1 (control) were cloned and expressed in accordance with Example 3. FIG. 12 is an SDS-PAGE evaluation of β17 fusion proteins and resulting purified polypeptide. The β17 polypeptide was fused C-terminally of NT_(D40K/K65D) (lane 1) or PGB1 (lane 2). Lane M denotes the size marker and the molecular weights are indicated to the left. Panel A shows an expression analysis before induction and after over-night expression at 20° C. in Bl21 E. coli cells. The NT_(D40K/K65D)-β17 fusion protein was expressed in E. coli at abundant levels exceeding those observed for β17 in fusion with PGB1. Panel B shows a solubility analysis after 2 min of sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (S) and insoluble (P) fractions. After sonication of harvested cells for 2 min followed by centrifugation, both fusion proteins were predominantly found in the soluble fraction.

Panel C shows fusion proteins after comparative Ni-Sepharose purification. Purification on Ni-Sepharose yielded 228 and 92 mg/L culture for β17 in fusion with NT_(D40K/K65D) and PGB1, respectively, and the amounts correlated to the intensities of the bands when analyzed with SDS-PAGE. The NT_(D40K/K65D) solubility tag was removed by proteolysis of a thrombin recognition sequence situated N-terminally of β17 followed by a second Ni-Sepharose purification step to separate the tag from the target protein. Panel D shows purification of β17 (SEQ ID NO: 27) from NT_(D40K/K65D)-β17 using Ni-sepharose chromatography and thrombin for cleavage and removal of the fusion tag. The lanes represent the supernatant after sonication (S), flow-through (FT), purified fusion protein (F), cleavage with thrombin (CL) and purified β17 target protein (T). The yield of soluble protein was 7.8 mg/L culture when measured immediately after purification.

In conclusion, NT_(D40K/K65D) as a solubility enhancing fusion tag for β17 enables an efficient purification process under non-denaturing conditions.

Example 9—Characterization of β17 Polypeptide (A) Gel Filtration

The hydrodynamic size of the β17 polypeptide (SEQ ID NO: 27) obtained in Example 8 was characterized using gel filtration. Gel filtration was performed on a 24 mL Superdex-200 column run at 0.3 mL/min. Samples were injected using a 200 uL loop and TBS, 5 mM EDTA, pH 8 with or without 150 mM NaCl as running buffer. The column was calibrated with apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa), that eluted at 10.25, 12.54, 13.65 and 16.18 mL, respectively.

FIG. 13 shows gel filtration of soluble NT_(D40K/K65D)-β17 fusion protein in running buffer without salt (FIG. 13A) and in running buffer supplemented with 150 mM NaCl (FIG. 13B). Analysis was performed after storage of the protein at −20° C. for one week (filled line) or at 4° C. for several days (dotted line).

The NT_(D40K/K65D) fusion protein remained highly soluble and migrated as a stable octamer after storage at −20° C. for one week or at 4° C. for several days when analyzed with gel filtration in the absence of salt (FIG. 13A). Similar results were obtained in the presence of 154 mM NaCl and 1 mM EDTA, but under these conditions the protein migrated as a stable dimer (FIG. 13B).

(B) ThT Assay

Aggregation kinetics were monitored using ThT fluorescence based on the enhanced quantum yield for ThT fluorescence as it binds to amyloid fibrils. Experiments were performed with 80 μM NT_(D40K/K65D)-β17 in 20 mM sodium phosphate buffer pH 8.0, 0.2 mM EDTA, with 10 μM ThT in microplate wells (Microplate Corning 3881, 96-well, low binding, half area, Corning Incorporated Life Sciences, Acton, Mass.). ThT fluorescence was recorded under quiescent conditions at 37° C., using a Fluostar Omega or Optima plate reader (BMG Labtech, Offenburg, Germany) with a 440 nm excitation filter and a 480 nm emission filter.

FIG. 14 shows β17 fibrillation after proteolysis of the fusion protein with thrombin. ThT fluorescence of the fusion protein in the presence of thrombin (1000:1) shows fibrillation of β17 as indicated by the increased ThT fluorescence over time (solid line). No fibrillation was observed for the fusion protein in absence of thrombin (dotted line). When purified in the absence of a fusion tag, β17 does not remain in solution long enough to study the fibrillation with ThT assay (data not shown). Here, we showed that this is feasible by keeping the protein in solution when fused to NT_(D40K/K65D), followed by proteolysis to release β17 at the start of the experiment.

Example 10—Expression of a Bri2 BRICHOS Domain Using a NT_(D40K/K65D) Fusion Protein

Bri2 is a TM glycoprotein composed of an N-terminal region followed by a TM domain, a linker region, a BRICHOS domain and a C-terminal region. The function is relatively unknown but the protein has been linked to Alzheimer's disease, Aβ precursor protein processing, Aβ homeostasis, apoptosis, tumor suppression and male reproduction. Mutations in Bri2 genes are associated with familial British dementia and familial Danish dementia, caused by the accumulation of amyloid fibrils in the brain. The situation is similar in Alzheimer's disease (AD) that is characterized by an accumulation of the amyloid beta peptide (Aβ), eventually forming brain plaques. The Bri2 BRICHOS domain is believed to act as an anti-amyloid chaperone, preventing amyloid formation and therefore is of interest as a promising therapeutic target.

The truncated BRICHOS domain Bri2₁₁₃₋₂₃₁ was expressed in fusion with NT_(D40K/K65D) or NT_(wt) (SEQ ID NO: 68-69, encoding SEQ ID NO. 66-67). The proteins were cloned and expressed essentially in accordance with Example 3, in Bl21 or Origami E. coli cells, at 20° C. over night or at 30° C. for 4 hours.

FIG. 15 is an SDS-PAGE evaluation of expression and solubility of Bri2 BRICHOS fusion proteins. The Bri2₁₁₃₋₂₃₁ domain was fused C-terminally of NT_(wt) (lane 1) or NT_(D40K/K65D) (lane 2). Lane M denotes the size marker and the molecular weights are indicated to the left. Panel A shows an expression analysis before induction (lane B) and after expression (lane A) at 20° C. over night (left gel) or at 30° C. for 4 hours (right gel) in Origami E. coli cells. Both fusion protein variants showed a high expression level in both strains of bacteria although NT_(wt) was slightly more efficient in Origami cells. Panel B shows a solubility analysis after lysozyme treatment followed by separation of soluble (lane S) and insoluble (lane P) fractions. Lysozyme treatment of harvested cells was inefficient although the NT_(D40K/K65D) fusion protein was slightly more soluble. Panel C shows solubility analysis after 2 min of sonication in 20 mM Tris-HCl, pH 8, followed by separation of soluble (lane S) and insoluble (lane P) fractions. After sonication, both fusion proteins were predominantly found in the soluble fraction. The analysis was performed on cells previously expressed at 20° C. over night (left gel) or at 30° C. for 4 hours (right gel).

The fusion proteins were purified on Ni-sepharose followed by thrombin cleavage and a second purification step to remove the tag. The final yield of Bri2₁₁₃₋₂₃₁ protein (SEQ ID NO: 50) was 55 mg/L culture in Origami and 12 mg/L culture in Bl21 using any NT variant.

Example 11—Characterization of Bri2-BRICHOS (A) SDS-PAGE Analysis of Purified Bri2-BRICHOS

FIG. 16 is an SDS-PAGE analysis of purification of Bri2-BRICHOS protein (SEQ ID NO: 50) obtained in Example 10. Samples from the purification steps were analyzed under reducing (panel A) and non-reducing (panel B) conditions for Bri2₁₁₃₋₂₃₁ in fusion with NT_(wt) (lane 1) or NT_(D40K/K65D) (lane 2). The molecular weights are indicated to the left. Supernatants were loaded on Ni-Sepharose and flow-through was collected (FT) followed by 4 washing steps with 10 mL running buffer (W1-W4). Pure fusion proteins were eluted with imidazole (F) and cleaved with thrombin (CL).

SDS-PAGE under reducing conditions (FIG. 16A) showed three bands (15, 12 and 10 kDa) for both fusion protein variants and they correlated well to the expected sizes of Bri2-BRICHOS (14 kDa) and NT (12 kDa and migrates as a smaller protein). The 12 kDa band is most probably truncated Bri2-BRICHOS unspecifically cleaved by thrombin. The same samples ran under non-reducing condition (FIG. 16B) showed that the 15 kDa and 12 kDa bands were able to form oligomers as expected from Bri2-BRICHOS.

(B) Gel Filtration

The conformation of the Bri2-BRICHOS protein (SEQ ID NO: 50) obtained in Example 10 was characterized using gel filtration essentially as set out in Example 9. Gel filtration was performed to estimate the proportion of fusion protein in their monomeric conformation. FIG. 17 shows gel filtration to determine the oligomeric state of Bri2₁₁₃₋₂₃₁ fusion proteins.

FIG. 17A shows a gel filtration analysis performed in 20 mM Tris, pH 8 for Bri2₁₁₃₋₂₃₁ in fusion with NT_(wt) (dotted line) or NT_(D40K/K65D) (filled line). When analyzing NT_(wt)-Bri2-BRICHOS, four peaks were distinguished. The peak at 25 kDa was close to the expected size of the monomeric fusion protein (29.7 kDa) but was small compared to other peaks. Dimer (50 kDa peak) and trimer (76 kDa peak) states were observed for both fusion proteins, and the NT_(wt) fusion protein also formed very large oligomers/aggregates (125 kDa peak). Interestingly, the amount of protein with a monomer conformation (25 kDa peak) was significantly higher with the NT_(D40K/K65D) fusion protein compared to the NT_(wt) fusion protein. The results indicate that the NT mutant increases proper folding of Bri2-BRICHOS, leading to larger fraction of soluble monomeric Bri2-BRICHOS.

FIG. 17B shows that purification of Bri2₁₁₃₋₂₃₁ without NT yields predominantly large oligomeric forms. Gel filtration analysis was performed to determine the oligomeric state of NT_(D40K/K65D)-Bri2-BRICHOS fusion protein compared to Bri2-BRICHOS produced in fusion with an S-tag, or Bri2-BRICHOS produced alone. Without fusion to the NT mutant, the proteins migrated close to the void volume and were either aggregated or in a state of very large oligomers.

The above data indicate that NT_(D40K/K65D) is able to mediate solubility and correct folding to Bri2-BRICHOS and also to prevent undesirable protein oligomerization.

Example 12—Expression of SP-A and SP-D Using a NT_(D40K/K65D) Fusion Protein

SP-A and SP-D are essential soluble innate immune proteins of the lung, which act to survey the lung and bind to pathogens leading to their neutralization, agglutination and clearance. They are also important modulators of the function of various immune cells and of the inflammatory immune response. Mice deficient in either SP-A or SP-D show increased susceptibility to pathogenic infections including RSV as well as exaggerated inflammatory responses after infectious bacterial challenge. Significant effort has been made to develop recombinant forms of SP-A and SP-D to understand their molecular action within the lung in models of human respiratory disease. Recombinant SP-A and SP-D may also have therapeutic potential for the treatment of various human lung diseases (Salgado, D. et al. Front Immunol 5, 623 (2014)).

The oligomerization of trimers increases the affinity of SP-A and SP-D for carbohydrates on the surface of pathogens. Smaller fragments with the ability to form trimers also show activity, but are less efficient compared the native form. Heterologous expression of full-length SP-A and SP-D has so far only been successful in mammalian systems while truncated forms have been expressed in bacterial and yeast systems.

Full-length hSP-A1 (UniProt ID: Q8IWL2), hSP-A2 (UniProt ID: Q8IWL1) and hSP-D (UniProt ID: P35247) as well as truncated fragments thereof: hSP-A1₈₁₋₂₂₈ and hSP-A2₈₁₋₂₂₈ (Silveyra, P. & Floros, J., Gene 531: 126-132 (2013); SEQ ID NO: 47-48), and hSP-D₂₀₄₋₃₅₅ (Hakansson, K. et al. Structure 7: 255-264 (1999); SEQ ID NO: 49) are expressed in fusion with NT_(D40K/K65D) (SEQ ID NO: 70-75, encoded by SEQ ID NO: 76-81). The fusion proteins are cloned and expressed essentially in accordance with Example 3, in Bl21 or Origami E. coli cells, at 20° C. over night or at 30° C. for 4 hours.

Example 13—Expression of Amyloid Beta (am Peptide Using a NT_(D40K/K65D) Fusion Protein

Aβ peptide is responsible for forming the amyloid plaques associated with Alzheimer's disease. The peptides are released from the amyloid precursor protein (APP) by proteolytic cleavage with beta and gamma secretase and can aggregate to form oligomers or larger fibrils that constitute amyloid plaques. The mechanisms of Aβ misfolding and fibrillation as well as the in vitro and in vivo toxicity of fibrils and intermediate oligomeric states have been extensively studied using synthetic peptides. Successful recombinant production of Aβ₁₋₄₀ and Aβ₁₋₄₂ peptides using a bacterial expression system has been demonstrated by extraction from inclusion bodies under denaturing conditions. This method does, however, not permit control over the oligomeric state of the peptides and require subsequent size-exclusion chromatography to obtain monomeric fractions prior to analysis.

To overcome this limitation Aβ1-42 (Uniprot ID P05067) is expressed in fusion with NT_(D40K/K65D) (SEQ ID NO: 82, encoded by SEQ ID NO: 83). The fusion protein is cloned and expressed essentially in accordance with Example 3, in Bl21 or Origami E. coli cells, at 20° C. over night or at 30° C. for 4 hours.

Example 14—Expression of Islet Amyloid Polypeptide (IAPP) Using a NT_(D40K/K65D) Fusion Protein

The islet amyloid polypeptide (amylin or IAPP; Uniprot ID P10997) is a peptide hormone that is co-secreted with insulin from pancreatic β-cells and has an important role in the regulation of blood glucose levels. Studies suggest that pancreatic amyloid formed by IAPP is associated with the development of type II diabetes. IAPP is expressed as a pro-peptide called ProIAPP, which is processed to IAPP upon stimulation. Human proIAPP has previously been expressed recombinantly in fusion with the Trx solubility tag and purified by extraction from inclusion bodies under denaturing conditions. Recombinant human IAPP (hIAPP) peptide is available from a number of commercial sources and is delivered in fusion with a solubility tag such as GST or in conjugation to BSA or OVA in order to keep the peptide soluble.

Human IAPP is expressed in fusion with NT_(D40K/K65D) (SEQ ID NO: 84, encoded by SEQ ID NO: 85). The fusion protein is cloned and expressed essentially in accordance with Example 3, in 8121 or Origami E. coli cells, at 20° C. over night or at 30° C. for 4 hours.

Example 15—Expression of hCAP18 Using a NT_(D40K/K65D) Fusion Protein

Cathelicidins are a family of antimicrobial and endotoxin-binding proteins found in granules of vertebrate neutrophils. Members of this family share a highly conserved 12 kDa N-terminus known as the cathelin-like domain. The biologically functional domain resides in the C-terminus and becomes activated when cleaved from the proprotein by serine proteases. The only human version of cathelicidin, hCAP18 is a proprotein to the antimicrobial peptide LL-37 that is released by extracellular cleavage with proteinase 3. hCAP18 inhibits growth of Gram-negative bacteria with efficiencies comparable to the mature peptide LL-37. However, recombinant production of hCAP18 is associated with low yields and an undetermined solubility, and new strategies are therefore needed for recombinant production.

hCAP18 without the signal peptide was cloned in fusion with NT_(wt) or NT_(D40K/K65D), respectively (SEQ ID NO: 86-87, encoded by SEQ ID NO: 88-89). Both fusion proteins were expressed in Origami E. coli cells at 20° C. overnight. FIG. 18 shows a SDS-PAGE analysis of expressed hCAP18 in fusion with N_(wt) or NT_(D40K/K65D) before induction (B) and after over-night expression (A) at 20° C. The arrow indicates the expected band corresponding to the protein of interest.

After sonication at 80% amplitude, 1 s pulses on/off, for 2 minutes in total, using non-denaturing amounts of urea (2 M), 0.7% Tween or 10% glycerol as buffer additives, the fusion proteins were predominantly found in the soluble fraction after centrifugation, independent of buffer additive. The fusion proteins were purified on Ni-sepharose with a final yield around 50 mg/L culture.

Example 16—Expression of Nicastrin Using a NT_(D40K/K65D) Fusion Protein

The γ-secretase protein complex is a four-component protease responsible for processing amyloid precursor protein (APP) and generating the Alzheimer's disease-associated peptide amyloid beta (Aβ). Modulation of the activity and specificity of γ-secretase represents a potential therapeutic strategy for the treatment of Alzheimer's disease. One of the components, nicastrin or NCT, is a type I transmembrane glycoprotein with a large extracellular domain (ECD), which is thought to play a critical role in the recruitment of γ-secretase substrates. Heterologous E. coli production of full-length nicastrin ECD has so far not been reported.

The ECD of human nicastrin (UniProt ID: Q92542) is expressed in fusion with NT_(D40K/K65D) (SEQ ID NO: 90, encoded by SEQ ID NO: 91). The fusion protein is cloned and expressed essentially in accordance with Example 3, in Bl21 or Origami E. coli cells, at 20° C. over night or at 30° C. for 4 hours.

Example 17—Expression of Green Fluorescent Protein (GFP) Using a NT_(D40K/K65D) Fusion Protein

GFP exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. GFP is frequently used as a reporter of expression due to the relatively small size that allows diffusion throughout cells without interfering with any biological processes. Many different mutants of GFP have been engineered and most importantly a S65T mutation dramatically increased the fluorescence and photostability. Enhanced GFP (eGFP) is the result of a F64L point mutation in addition to the S65T mutation, that showed increased folding efficiency at 37° C. and allowed practical use of GFP in mammalian cells

eGFP was cloned in fusion with NT_(wt) and NT_(D40K/K65D) (SEQ ID NO: 92-93, encoded by SEQ ID NO: 94-95) and expressed in Bl21 E. coli cells at 20° C. over night. The cells were disrupted by lysozyme treatment instead of sonication. This method is less effective to solubilize protein but was required to maintain GFP fluorescence, which is otherwise partly lost during sonication treatment. Still, around 30-40% of the fusion protein was found in the soluble fraction after centrifugation and most protein was recovered during Ni-sepharose purification.

Example 18—NT_(D40K/K65D) in Fusion with rSP-C33Leu Arranges into Micelle-Like Particles

A purified and soluble NT_(D40K/K65D)-SP-C33Leu fusion protein (SEQ ID NO 56) was obtained as set out in Examples 3-4 and subjected to size exclusion chromatography (SEC) and transmission electron microscopy (TEM). For TEM, a purified and soluble NT_(D40K/K65D) protein (SEQ ID NO: 2) was used as control.

Size-Exclusion Chromatography

Purified fusion protein was diluted to 2 mg/mL in running buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8.0). A Superdex 200 column was equilibrated in running buffer and 200 uL of the sample was run through the column at a rate of 0.5 mL/min. Elution of protein was detected by measuring optical absorbance at 280 nm. Molecular weight standards ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) (GE Healthcare) were run and eluted at 8.56 mL, 10.65 mL, 12.06 mL, 12.96 mL, 14.26 mL and 15.64 mL, respectively.

Transmission Electron Microscopy

The samples were diluted in 20 mM Tris, pH 8. For negative staining, 3 μl samples were applied to glow-discharged carbon-coated copper grids, stained with 2% (w/v) uranyl acetate and air-dried. The grids were checked using JEOL JEM-2100f transmission electron microscope operated at 200 kV. Images were collected with TVIPS TemCam-F415 4k×4k CCD-camera (Tietz Video and Image Processing Systems GmbH, Gauting, Germany) using a nominal magnification of 60000.

While the NT_(D40K/K65D)-SP-C33Leu monomer has a calculated molecular mass of of 19 kDa, the SEC analysis presented in FIG. 19 showed a well-defined oligomer population with an estimated size of 510 kDa, corresponding to particles with a hydrodynamic radius around 10 nm. The presence of such micelle-like particles with a size of 10-15 nm was confirmed using negative staining transmission electron microscopy (TEM). In FIG. 20, panel (a) TEM of negatively stained NT_(D40K/K65D)-SP-C33Leu fusion protein shows 10-15 nm sized particles. In panel (b), TEM of negatively stained NT_(D40K/K65D) protein at the same concentration served as negative control. No particles were observed for the NT_(D40K/K65D) protein alone.

Example 19 (Comparative)—Protein Expression of Fusion Proteins Comprising NT_(wt) and NT_(A72R)

EP 2 644 619 A1 discloses the solubility-enhancing moiety NT_(A72R) and fusion proteins comprising the same. NT_(A72R) is a constitutive monomer also below a pH of 6.4. The fusion proteins NT_(A72R)-SP-C33Leu (SEQ ID NO: 100) and NT_(wt)-SP-C33Leu (SEQ ID NO: 57) were expressed in E. coli BL21 (DE3) cells and purified in accordance with Examples 3-4.

The obtained proteins were separated by SDS-PAGE and stained with Coomassie. The different fusion proteins were investigated and the expression (production) levels per bacterium were found to be in the order NT_(wt)-SP-C33Leu>NT_(A72R)-SP-C33Leu.

In conclusion, NT_(A72R) does not improve the fusion protein expression levels compared to NT_(wt).

Example 20—Expression and Purification of Modified Spidroin Proteins

A spidroin protein with a modified fibronectin-derived RGD loop, FN_(cc), a repetitive moiety and a CT moiety from a minor ampullate spidroin (MiSp) was cloned in fusion with NT_(D40K/K65D) and Z, respectively (SEQ ID NO: 101-102). Identical fusion proteins but with a CT moiety from a major ampullate spidroin (MaSp) were also cloned (SEQ ID NO: 103-104).

To test expression levels of the modified spidroin proteins, the fusion proteins were cloned into pT7 expression vectors and transformed into chemically competent E. coli BL21 (DE3) cells.

Plasmid-containing cells were inoculated to 150 mL LB medium with 50 mg/L kanamycin and grown at 30° C. and 220 rpm over night. 5 mL over-night culture was inoculated to 500 mL LB medium (1/100) with kanamycin and cells were further grown at 30° C. to OD ˜1. The cells were induced by addition of IPTG to a final concentration of 0.3 mM and expression was performed at 15° C. over night. After 19 hours of expression the cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8 to 40 mL and stored at −20° C. for at least 24 hours. Cells were solubilized by the addition of lysozyme. The soluble and insoluble fractions were separated by centrifugation at 21612×g, 4° C. for 30 min.

Duplicate culture samples equal to OD₆₀₀=1 were taken after 4 and 19 hours of induction during expression. Samples were pelleted through centrifugation at 13000×g, 4° C. for 10 min, the supernatant was discarded and the pellets were stored at −20° C. for at least 24 h. Pelleted material were lysed using CelLytic B™ with the addition of lysozyme. One sample per time point was used for the analysis of the soluble and insoluble fraction which were separated through centrifugation at 13000×g, RT for 10 min. The other samples were used for whole cell analysis without separating the soluble and insoluble fractions.

To the samples equal amounts of reducing SDS-PAGE loading buffer was added, boiled for 5 min at 95° C. and subsequently loaded in equal amounts on a 12% acrylamide gel. The proteins were transferred to a membrane using iBlot 2® Dry Blotting System. iBind™ Western Device is used for blotting using an IRDYE800CW® fluorophore labeled chicken anti-his antibody. Detection is performed using a Liquor Odyssey fc® imaging system.

To the clear lysate from the cultures, NaCl and imidazole was added to a final concentration of 500 mM and 20 mM respectively and loaded to an IMAC column, previously packed with Zn-Sepharose (GE Healthcare) and equilibrated with loading buffer (20 mM Tris, 20 mM Imidazole and 500 mM NaCl, pH 8). Bound protein was washed with 20 mM Tris-HCl, 49 mM imidazole, 500 mM NaCl, pH 8 and eluted with 20 mM Tris-HCl, 220 mM imidazole, 500 mM NaCl, pH 8 in 1 mL fractions. The absorbance at 280 nm was measured for each fraction, and protein-rich fractions were pooled. Imidazole was removed by over-night dialysis at 4° C. and in 5 L loading buffer, using a Spectra/Por® dialysis membrane with a 6-8 kDa molecular weight cut-off. The purity of the protein in each step was determined by SDS-PAGE using a 12% acrylamide gel stained with Coomassie Brilliant Blue.

After storage at −20° C., the fusion proteins were thawed at 4° C., centrifuged for 10 min at 4570×g and concentrated. Samples were stored at 4° C. The stability was evaluated numerically by measuring concentration over the span of 3 days using Nano Drop™ and visually by SDS-PAGE, using a 12% acrylamide gel stained with InstantBlue™ protein stain. It is concluded that NT_(D40K/K65D) is more effective than the Z-tag in keeping highly aggregation-prone purified spidroin proteins in solution.

Example 21—Expression of Spidroin Fusion Proteins

To test expression levels of spidroin fusion proteins, the IgG fragment sCD40 covalently linked to 4RepCT was cloned in fusion with NT_(D40K/K65D) and Z, respectively (SEQ ID NO: 105-106), into pT7 expression vectors and transformed into chemically competent E. coli BL21 (DE3) cells.

Plasmid-containing cells were inoculated to 150 mL LB medium with 50 mg/L kanamycin and grown at 30° C. and 220 rpm over night. 5 mL over-night culture was inoculated to 500 mL LB medium (1/100) with kanamycin and cells were further grown at 30° C. to OD ˜1. The cells were induced by addition of IPTG to a final concentration of 0.3 mM and expression was performed at 15° C. over night. After 19 hours of expression the cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8 to 40 mL and stored at −20° C. for at least 24 hours.

In order to analyse the amount of protein expressed in the soluble and insoluble fraction, respectively, culture samples are taken and analyzed using western blot, as described in Example 20.

Example 22—Expression of IgG Fragments

The IgG fragment sCD40 covalently linked to a sortase recognition sequence in fusion with NT_(D40K/K65D) and Z, respectively (SEQ ID NO: 107-108), were cloned into pT7 expression vectors and transformed into chemically competent E. coli BL21 (DE3) cells.

Plasmid-containing cells were inoculated to 20 mL LB medium with 50 mg/L kanamycin and grown at 30° C. and 220 rpm over night. 5 mL over-night culture was inoculated to 500 mL LB medium (1/100) with kanamycin and cells were further grown at 30° C. to OD ˜1. The cells were induced by addition of IPTG to a final concentration of 0.3 mM, and expression was performed at 15° C. over night. After 19 hours of expression the cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 8 to 40 mL and stored at −20° C. for at least 24 hours.

In order to analyse the amount of protein expressed in the soluble and insoluble fraction, respectively, culture samples are taken and analyzed using western blot, as described in Example 20. 

1. A method for producing a desired protein or polypeptide, comprising: a) expressing in a suitable host a fusion protein comprising a first moiety which is the desired protein or polypeptide and a second moiety that enhances the solubility of the first moiety; wherein the second moiety comprises 100-160 amino acid residues having at least 70% identity with any one of SEQ ID NOs: 1, 13-23, 25-26, and 96-99, wherein the amino acid residue corresponding to position 40 in SEQ ID NO:1 is Lys, Arg or His; and wherein the amino acid residue corresponding to position 65 in SEQ ID NO:1 is Asp or Glu; and b) obtaining a composition comprising the fusion protein.
 2. The method according to claim 1, further comprising: b1) isolating the fusion protein.
 3. The method according to claim 2, wherein step b1) of isolating the fusion protein comprises: b1a) precipitation of the fusion protein; and b1b) suspending the precipitated fusion protein in an aqueous solvent, wherein the fusion protein is soluble in the aqueous solvent.
 4. The method according to claim 3, wherein the precipitation step comprises salting out at high salt concentration.
 5. The method according to claim 1 further comprising: c) cleaving the fusion protein to release the desired protein or polypeptide from the second moiety as cleavage products.
 6. The method according to claim 5, further comprising: c1) isolating the desired protein or polypeptide.
 7. The method according to claim 6, wherein step c1) of isolating the desired protein or polypeptide comprises: c1b) extracting the desired protein or polypeptide by suspending the cleavage products in an organic solvent; wherein the desired protein or polypeptide is soluble in the organic solvent; and wherein the second moiety is not soluble in the organic solvent.
 8. The method according to claim 7, wherein the organic solvent of step c1b) comprises a lower alkyl alcohol.
 9. The method according to claim 7, wherein step c1) of isolating the desired protein or polypeptide further comprises, prior to the extraction step c1b), the following step: c1a) precipitation of the cleavage products.
 10. The method according to claim 9, wherein the precipitation step comprises salting out at high salt concentration.
 11. The method according to claim 1, further comprising: b1) isolating the fusion protein; c) cleaving the fusion protein to release the desired protein or polypeptide from the second moiety as cleavage products; and c1) isolating the desired protein or polypeptide.
 12. The method according to claim 11, with the proviso that the method does not contain any separation step involving gel filtration, chromatography or any other solid phase adsorption-based separation.
 13. The method according to claim 1, wherein the composition obtained in step b) comprises micelles of the fusion protein.
 14. The method according to claim 1, wherein the second moiety has at least 70% identity with any one of SEQ ID NO:1 and 13-21.
 15. The method according to claim 1, wherein the second moiety has at least 70% identity with SEQ ID NO:1.
 16. The method according to claim 1, wherein the amino acid residue corresponding to position 40 in SEQ ID NO:1 is Lys or Arg.
 17. The method according to claim 16, wherein the amino acid residue corresponding to position 40 in SEQ ID NO:1 is Lys.
 18. The method according to claim 1, wherein the amino acid residue corresponding to position 65 in SEQ ID NO:1 is Asp.
 19. The method according to claim 1, wherein at least one cleavage site is present between the first moiety and the second moiety of the fusion protein. 